Antibody against trimeric TNFα complex

ABSTRACT

It has been demonstrated that certain compounds bind to TNF and stabilise a conformation of trimeric TNF that binds to the TNF receptor. Antibodies which selectively bind to complexes of such compounds with TNF superfamily members are disclosed. These antibodies may be used to detect further compounds with the same activity, and as target engagement biomarker.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (1089-0008US1_SL.txt;Size: 44 KB; and Date of Creation Jun. 17, 2019) is herein incorporatedby reference in its entirety.

FIELD OF THE INVENTION

This invention relates to antibodies, particularly antibodiesrecognising a specific epitope, which may be used to screen for smallmolecule modulators of TNFα. The invention relates to antibodies whichselectively bind to such TNFα small molecule modulator complexes, anduses of such antibodies. The present invention also relates to assaysfor identifying new modulators of TNFα using said antibodies.

BACKGROUND OF THE INVENTION

The Tumour Necrosis Factor (TNF) superfamily is a family of proteinsthat share a primary function of regulating cell survival and celldeath. Members of the TNF superfamily share a common core motif, whichconsists of two antiparallel β-pleated sheets with antiparallelβ-strands, forming a “jelly roll” β-structure. Another common featureshared by members of the TNF superfamily is the formation of homo- orheterotrimeric complexes. It is these trimeric forms of the TNFsuperfamily members that bind to, and activate, specific TNF superfamilyreceptors.

TNFα is the archetypal member of the TNF superfamily. Dysregulation ofTNFα production has been implicated in a number of pathologicalconditions of significant medical importance. For example, TNFα has beenimplicated in rheumatoid arthritis, inflammatory bowel diseases(including Crohn's disease), psoriasis, Alzheimer's disease (AD),Parkinson's disease (PD), pain, epilepsy, osteoporosis, asthma, systemiclupus erythematosus (SLE) and multiple sclerosis (MS). Other members ofthe TNF superfamily have also been implicated in pathologicalconditions, including autoimmune disease.

Conventional antagonists of TNF superfamily members are macromolecularand act by inhibiting the binding of the TNF superfamily member to itsreceptor. Examples of conventional antagonists include anti-TNFαantibodies, particularly monoclonal antibodies, such as infliximab(Remicade®), adalimumab (Humira®) and certolizumab pegol (Cimzia®), orsoluble TNFα receptor fusion proteins, such as etanercept (Enbrel®).

SUMMARY OF THE INVENTION

The present inventors have identified classes of small molecularentities (SME) that modulate TNFα. These compounds act by binding to thehomotrimeric form of TNFα, and inducing and/or stabilising aconformational change in the homotrimer of TNFα. For example,homotrimers of TNFα with the compound bound can bind to TNFα receptors,but are less able, or unable, to initiate signalling downstream of theTNFα receptor. These compounds can be used in the treatment ofconditions mediated by TNFα.

The present inventors have developed antibodies that bind selectively tocomplexes comprising such compounds and TNFα. These antibodies may beused to identify further compounds that are capable of inhibiting TNFαin this manner, and may also be used as target engagement biomarkers.

Accordingly, the present invention provides an antibody which binds toan epitope comprising at least the following residues of trimeric TNFα:

(a) Y115

(b) Q149; and

(c) N137 or K98 or both,

wherein Y115 and Q149 are present on the C chain of trimeric TNFα, andN137 and K98 are present on the A chain of trimeric TNFα, and whereinthe residue numbering is according to SEQ ID NO: 36.

The present invention also provides:

-   -   An antibody which competes for binding to TNFα with an antibody        as defined above.    -   An isolated polynucleotide encoding an antibody of the        invention.    -   An antibody of the invention for use in a method of treatment of        the human or animal body by therapy.    -   A pharmaceutical composition comprising an antibody of the        invention and a pharmaceutically acceptable adjuvant and/or        carrier.    -   Use of an antibody of the invention as a target engagement        biomarker for the detection of a compound-trimer complex in a        sample obtained from a subject; wherein said antibody is        detectable and said complex comprises trimeric TNFα and a        compound that is capable of binding to trimeric TNFα, whereby        the compound-trimer complex binds to the requisite receptor and        modulates the signalling induced by the trimer through the        receptor.    -   A method of detecting target engagement of a compound to        trimeric TNFα, whereby the compound-trimer complex binds to the        requisite receptor and modulates the signalling induced by the        trimer through the receptor, said method comprising:        -   (a) obtaining a sample from a subject administered said            compound;        -   (b) contacting an antibody of the invention to said sample            and a control sample, wherein said antibody is detectable;        -   (c) determining the amount of binding of said detectable            antibody to said sample and said control sample,    -   wherein binding of said detectable antibody to said sample        greater than binding of said detectable antibody to said control        sample indicates target engagement of said compound to said        trimeric TNFα.    -   Use of an antibody of the invention in screening for a compound        that elicits a conformational change in a TNFα trimer, wherein        said conformational change modulates the signalling of the        requisite receptor on binding of the trimeric TNFα.    -   A complex comprising a TNFα trimer and a compound that is bound        thereto, whereby the compound-trimer complex binds to the        requisite receptor and modulates the signalling induced by the        trimer through the receptor, wherein said complex binds to an        antibody of the invention with a K_(D-ab) of 1 nM or less.    -   A compound that is capable of binding to a TNFα trimer to form a        complex, whereby the compound-trimer complex binds to the        requisite receptor and modulates the signalling induced by the        trimer through the receptor, wherein the compound-trimer complex        binds to an antibody of the invention with a K_(D-ab) of 1 nM or        less.    -   A method of identifying a compound that is capable of binding to        a TNFα trimer and modulating signalling of the trimer through        the requisite receptor, comprising the steps of:    -   (a) performing a binding assay to measure the binding affinity        of a test compound-trimer complex comprising a TNFα trimer and a        test compound to an antibody of the invention that selectively        binds to said complex;    -   (b) comparing the binding affinity as measured in step (a) with        the binding affinity of a different compound-trimer complex        known to bind with high affinity to the antibody referred to in        step (a); and    -   (c) selecting the compound present in the compound-trimer        complex of step (a) if its measured binding affinity is        acceptable when considered in the light of the comparison        referred to in step (b).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 highlights residues N168, I194, F220 and A221 on the crystalstructure of human TNFα.

FIG. 2 shows results of HPLC experiments with the CA185_01974 mFab andcompound (1). Peaks corresponding to excess Fab appear at a 1.5× and2.0× excess. The stoichiometry was therefore determined to be 1 Fab: 1TNFα trimer.

FIG. 3 shows results of HPLC experiments with the CA185_01979 mFab andcompound (1). Again, peaks corresponding to excess Fab appear at a 1.5×and 2.0× excess. The stoichiometry was therefore also determined to be 1Fab: 1 TNFα trimer.

FIG. 4 presents results of total TNFα ELISA with compounds (3), (4) and(5) using a commercial anti-TNFα polyclonal antibody.

FIG. 5 presents results of conformation specific TNFα ELISA withCA185_01974.0 and compounds (3), (4) and (5). Apo TNFα gave no signal inthis assay, demonstrating the specific nature of the binding of antibodyCA185_01974 to compound-bound TNFα.

FIG. 6 shows FACS histogram plots of staining with CA185_01974 andCA185_01979 at 1 and 10 μg/ml. These plots demonstrate that theantibodies only recognise TNFα which has been pre-incubated withcompound (1). There is no staining with the DMSO control.

FIG. 7 shows FACS histogram plots of staining with CA185_01974 for aparental NS0 cell line and an engineered NS0 cell line, whichoverexpresses membrane TNFα. Cells were incubated with compound (1) orDMSO and stained with the antibody Fab fragment. Again, results indicateno staining for the DMSO control (for either the parental or engineeredcell line). In the presence of compound (1) staining is, however,observed for the engineered cell line.

FIG. 8 shows sensograms for the determination of affinity values forCA185_01974 using cynomolgus TNFα. Controls (top panels) containedcynomolgus TNFα and DMSO. The bottom panels then present duplicatedexperiments for cynomolgus TNFα complexed with compound (4).

FIG. 9 shows sensograms for the determination of affinity values forCA185_01974 using human TNFα. Controls (top panels) contained human TNFαand DMSO. The bottom panels then present duplicated experiments forhuman TNFα complexed with compound (4).

FIG. 10 shows the structures of compounds (1)-(7).

FIG. 11 shows the structure of the Fab1974 bound to a complex of humanTNFα asymmetric trimer, compound (2) and 2 human TNFR1 receptors.

FIG. 12 shows the same structure as FIG. 11, but with a symmetricaltrimer (i.e. in the absence of compound) modelled in computationally.With this symmetric trimer, the A chain of the trimer retains theinteraction with the Fab fragment. However, there is a steric clashbetween the Fab fragment and the C chain of the trimer.

FIG. 13 shows as spheres the experimentally determined epitope on the Aand C chains of the TNFα trimer with CA185_1974.

FIG. 14 shows that CA185_1974 represents a suitable reagent formeasuring target occupancy in a complex biological matrix. CA185_1974was used as capture reagent to measure TNFα complexed with compound (2)using a high sensitivity ELISA, with a commercial anti-TNFα antibody asthe detection reagent. TNFα pre-incubated with either excess compound ofDMSO was spiked into neat human plasma (depleted or endogenous TNFα) anddiluted to various concentrations. Signal generated by TNFα-smallmolecule inhibitor complex clearly increased proportionally with TNFαspike concentration. There was no significant detection of TNFαpre-incubate with DMSO (apo-TNF) at any TNFα spike concentrationstested. The TNFα-small molecule complex signal was significantly higherthan apo-TNFα background with probability of at least 0.999 at TNFαconcentrations of 25 pg/ml and above.

FIG. 15 shows single cycle kinetics of compound (1) binding to humanTNFα directly immobilised (a) or via capture with CA185_1974 (b).Kinetics change from “slow on-slow off” with directly immobilisedapo-TNF to “fast on-fast off” when captured via theconformation-selective antibody CA1974.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows the LCDR1 of CA185_01974.0.

SEQ ID NO: 2 shows the LCDR2 of CA185_01974.0.

SEQ ID NO: 3 shows the LCDR3 of CA185_01974.0.

SEQ ID NO: 4 shows the HCDR1 of CA185_01974.0.

SEQ ID NO: 5 shows the HCDR2 of CA185_01974.0.

SEQ ID NO: 6 shows the HCDR3 of CA185_01974.0.

SEQ ID NO: 7 shows the amino acid sequence of the LCVR of CA185_01974.0.

SEQ ID NO: 8 shows the amino acid sequence of the HCVR of CA185_01974.0.

SEQ ID NO: 9 shows the DNA sequence of the LCVR of CA185_01974.0.

SEQ ID NO: 10 shows the DNA sequence of the HCVR of CA185_01974.0.

SEQ ID NO: 11 shows the amino acid sequence of the kappa light chain ofCA185_01974.0.

SEQ ID NO: 12 shows the amino acid sequence of the mIgG1 heavy chain ofCA185_01974.0.

SEQ ID NO: 13 shows the amino acid sequence of the mFab (no hinge) heavychain of CA185_01974.0.

SEQ ID NO: 14 shows the DNA sequence of the kappa light chain ofCA185_01974.0.

SEQ ID NO: 15 shows the DNA sequence of the mIgG1 heavy chain ofCA185_01974.0.

SEQ ID NO: 16 shows the DNA sequence of the mFab (no hinge) heavy chainof CA185_01974.0.

SEQ ID NO: 17 shows the LCDR2 of CA185_01979.0.

SEQ ID NO: 18 shows the LCDR3 of CA185_01979.0.

SEQ ID NO: 19 shows the HCDR1 of CA185_01979.0.

SEQ ID NO: 20 shows the HCDR2 of CA185_01979.0.

SEQ ID NO: 21 shows the HCDR3 of CA185_01979.0.

SEQ ID NO: 22 shows the amino acid sequence of the LCVR ofCA185_01979.0.

SEQ ID NO: 23 shows the amino acid sequence of the HCVR ofCA185_01979.0.

SEQ ID NO: 24 shows the DNA sequence of the LCVR of CA185_01979.0.

SEQ ID NO: 25 shows the DNA sequence of the HCVR of CA185_01979.0.

SEQ ID NO: 26 shows the amino acid sequence of the kappa light chain ofCA185_01979.0.

SEQ ID NO: 27 shows the amino acid sequence of the mIgG1 heavy chain ofCA185_01979.0.

SEQ ID NO: 28 shows the amino acid sequence of the mFab (no hinge) heavychain of CA185_01979.0.

SEQ ID NO: 29 shows the DNA sequence of the kappa light chain ofCA185_01979.0.

SEQ ID NO: 30 shows the DNA sequence of the mIgG1 heavy chain ofCA185_01979.0.

SEQ ID NO: 31 shows the DNA sequence of the mFab (no hinge) heavy chainof CA185_01979.0.

SEQ ID NO: 32 shows the amino acid sequence of rat TNFα.

SEQ ID NO: 33 shows the amino acid sequence of mouse TNFα.

SEQ ID NO: 34 shows the amino acid sequence of human TNFα.

SEQ ID NO: 35 shows the amino acid sequence of the soluble form of humanTNFα.

SEQ ID NO: 36 shows the amino acid sequence of the soluble form of humanTNFα, but without the initial “S” (which is a cloning artefact in SEQ IDNO: 35).

SEQ ID NO: 37 shows the amino acid sequence of the extracellular domainof human TNFR1 with N54D and C182S mutations.

SEQ ID NO: 38 shows the amino acid sequence of SEQ ID NO: 37, butwithout the initial “G”, which is a cloning artefact.

DETAILED DESCRIPTION OF THE INVENTION

Modulators of TNF Superfamily Members

Test compounds that bind to trimeric forms of TNF superfamily membershave been identified. The following disclosure relates generally tobinding of these compounds to any TNF superfamily member. An embodimentof the invention relates specifically to binding of such compounds toTNFα.

The test compounds are small molecular entities (SMEs) that have amolecular weight of 1000 Da or less, 750 Da or less, or 600 Da or less.The molecular weight may be in the range of about 50-about 1000 Da orabout 100-about 1000 Da. These compounds stabilise a conformation of thetrimeric TNF superfamily member that binds to the requisite TNFsuperfamily receptor and modulates the signalling of the receptor.Examples of such compounds include compounds of formulae (1)-(7).

The stabilising effect of compounds on trimeric forms of TNF superfamilymembers may be quantified by measuring the thermal transition midpoint(Tm) of the trimers in the presence and absence of the compound. Tmsignifies the temperature at which 50% of the biomolecules are unfolded.Compounds which stabilise TNF superfamily member trimers will increasethe Tm of the trimers. Tm may be determined using any appropriatetechnique known in the art, for example using differential scanningcalorimetry (DSC) or fluorescence probed thermal denaturation assays.

The compounds may bind inside the central space present within the TNFsuperfamily member trimer (i.e. the core of the trimer).

These compounds may turn the TNF superfamily member into a TNFsuperfamily receptor antagonist. These compounds are therefore capableof blocking the TNF superfamily member signalling without having tocompete with the high affinity interaction between the TNF superfamilymember and its receptor.

Alternatively, the compounds may stabilise a conformation of thetrimeric TNF superfamily member that binds to the requisite TNFsuperfamily receptor and enhances the signalling of the receptor. Thesecompounds are therefore capable of increasing the TNF superfamily membersignalling without having to compete with the high affinity interactionbetween the TNF superfamily member and its receptor.

Where herein the compounds are described as antagonists, it will beunderstood that the compounds may equally be agonists and increasesignalling by a TNF superfamily receptor that is bound to a complex of aTNF superfamily member trimer and such an agonist compound. Similarly,where other disclosure refers to antagonistic compounds, methods ofidentifying such compounds and uses of such compounds, this disclosuremay refer equally to agonist compounds.

The compounds described herein are allosteric modulators that bind tothe natural agonists of the TNF superfamily receptors, i.e. to trimericforms of TNF superfamily members and drive these trimers to adopt aconformation that still binds to the requisite TNF superfamily receptorand modulates signalling by the receptor. By modulating, it will beunderstood that the compound may have an antagonistic effect and sodecrease signalling by a TNF superfamily receptor, or else a stimulatoryeffect and so increase or enhance signalling by a TNF superfamilyreceptor.

These compounds may convert the natural TNF superfamily member agonistsinto antagonists. In contrast, conventional TNF superfamily memberantagonists bind to the TNF superfamily member or the TNF superfamilyreceptor and prevent the binding of the TNF superfamily member to therequisite receptor. In the alternative, the compounds may increasesignalling by a TNF superfamily receptor when the TNF superfamily memberis bound compared to the level of signalling by the TNF superfamilyreceptor when the TNF superfamily member is bound in the absence of thecompound. The compounds may therefore convert the natural TNFsuperfamily member agonists into so-called “super-agonists”. Thecompounds may therefore also be known as allosteric modulators of ligandactivity (AMLAs).

The compounds are not limited in terms of their chemical formula orstructure, provided that they bind to at least one TNF superfamilymember and stabilise a conformation of the trimeric TNF superfamilymember that binds to the requisite TNF superfamily receptor andmodulates the signalling of the TNF superfamily receptor. The compoundscan therefore be identified using the antibodies and methods describedherein. A non-limiting example is compounds that may comprise abenzimidazole moiety or an isostere thereof.

The compounds may increase the binding affinity of TNF superfamilymembers (in the form of a compound-trimer complex) to the requisitereceptor compared to the binding affinity of the TNF superfamily membersto the requisite receptor in the absence of the compounds.

The compounds bind to the trimeric forms of TNF superfamily members.Such compounds may bind specifically (or selectively) to the trimericforms of one or more TNF superfamily members. A compound may bindspecifically (or selectively) to only one of the TNF superfamilymembers, but not to any other TNF superfamily members. A compound mayalso bind specifically to two, three, four or up to all of the TNFsuperfamily members. By specific (or selective), it will be understoodthat the compounds bind to the molecule or molecules of interest, inthis case the trimeric form of the TNF superfamily member, with nosignificant cross-reactivity to any other molecule, which may includeother members of the TNF superfamily. Cross-reactivity may be assessedby any suitable method, for example surface plasmon resonance.Cross-reactivity of a compound for the trimeric form of a TNFsuperfamily member with a molecule other than the trimeric form of thatparticular TNF superfamily member may be considered significant if thecompound binds to the other molecule at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or 100% as strongly as it binds to the trimeric form of the TNFsuperfamily member of interest. For example, cross reactivity may beconsidered significant if the compound binds to the other molecule about5%-about 100%, typically about 20%-about 100%, or about 50%-about 100%as strongly as it binds to the trimeric form of the TNF superfamilymember of interest. A compound that is specific (or selective) for thetrimeric form of a TNF superfamily member may bind to another moleculeat less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%,45%, 40%, 35%, 30%, 25% or 20% the strength that it binds to thetrimeric form of the TNF superfamily member (down to zero binding). Thecompound may bind to the other molecule at less than about 20%, lessthan about 15%, less than about 10% or less than about 5%, less thanabout 2% or less than about 1% the strength that it binds to thetrimeric form of the TNF superfamily member (down to zero binding).

The rates at which a test compound binds to a TNF superfamily member isreferred to herein as the “on” rate” k_(on-c) and the rate at which thetest compound dissociates from the TNF superfamily member is referred toherein as the “off” rate or k_(off-c). As used herein, the symbol“K_(D-c)” denotes the binding affinity (dissociation constant) of a testcompound for a TNF superfamily member. K_(D-c) is defined ask_(off-c)/k_(on-c). Test compounds may have slow “on” rates, which canbe measured in minutes by mass spectral analysis of the TNF superfamilymember and compound-trimer complex peak intensities. K_(D-c) values fora test compound can be estimated by repeating this measurement atdifferent TNF superfamily member: compound-trimer complex ratios. As anon limiting example, binding of compounds to TNF superfamily trimerscan be characterized by fast “on” rates, about 10⁷ M⁻¹s⁻¹, with slow“off” rate, for example values of 10⁻³ s⁻¹, 10⁻⁴ s⁻¹, or no measurable“off” rate.

As used herein, the symbol “k_(on-r)” denotes the rate (the “on” rate)at which a compound-trimer complex binds to a TNF superfamily receptor.As used herein, the symbol “k_(off-r)” denotes the rate (the “off” rate)at which a compound-trimer complex dissociates from a TNF superfamilyreceptor. As used herein, the symbol “K_(D-r)” denotes the bindingaffinity (dissociation constant) of a compound-trimer complex for asuperfamily receptor. K_(D-r) is defined as k_(off-r)/k_(on-r).

The K_(D-r) value of the TNF superfamily member for binding to itsreceptor in the presence of the test compound (i.e. in the form of acompound-trimer complex) may be at least about 1.5 times, 2 times, 3times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50times, 60 times, 70 times, 80 times, 90 times, 100 times lower than theK_(D-r) value of the TNF superfamily member for binding to its receptorin the absence of the test compound. The K_(D-r) value of thecompound-trimer complex for binding to the TNF superfamily member may bedecreased at least about 1.5 times, or at least about 3 times, or atleast about 4 times the K_(D-r) value of the TNF superfamily trimerbinding to the TNF superfamily receptor in the absence of the testcompound, i.e. the binding affinity of the compound-trimer complex forthe TNF superfamily receptor may be increased at least about 1.5-fold,at least about three-fold, at least about four-fold compared to thebinding affinity of the TNF superfamily trimer to the TNF superfamilyreceptor in the absence of test compound.

A compound described herein may increase the binding affinity of the TNFsuperfamily member to its receptor by about 2 times, 3 times, 4 times, 5times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70times, 80 times, 90 times, 100 times or more compared to the bindingaffinity of the TNF superfamily member to its receptor in the absence ofthe compound.

The binding affinity may be given in terms of binding affinities(K_(D-r)) and may be given in any appropriate units, such as μM, nM orpM. The smaller the K_(D-r) value, the larger the binding affinity ofthe TNF superfamily member to its receptor.

The K_(D-r) value of the TNF superfamily member for binding to itsreceptor in the presence of the compound may be at least about 1.5times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times,40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 timeslower than the K_(D-r) value of the TNF superfamily member for bindingto its receptor in the absence of the test compound.

The decrease in the K_(D-r) value of the compound-trimer complex forbinding to the TNF superfamily receptor compared to the K_(D-r) value ofthe TNF superfamily trimer alone binding to the TNF superfamily receptormay result from an increase in the on rate (k_(on-r)) of thecompound-trimer complex binding to the TNF superfamily receptor comparedto the TNF superfamily trimer alone, and/or a decrease in the off rate(k_(off-r)) compared to the TNF superfamily trimer alone. The on rate(k_(on-r)) of the compound-trimer complex binding to the TNF superfamilyreceptor is generally increased compared to the TNF superfamily trimeralone. The off rate (k_(off-r)) of the compound-trimer complex bindingto the TNF superfamily receptor is generally decreased compared to theTNF superfamily trimer alone. Most suitably, the on rate (k_(on-r)) ofthe compound-trimer complex binding to the TNF superfamily receptor isincreased, and the off-rate (k_(off-r)) of the compound-trimer complexbinding to the TNF superfamily receptor is decreased, compared to theTNF superfamily trimer alone. The k_(on-r) value of the compound-trimercomplex to the requisite TNF superfamily receptor may be increased by atleast about 1.5-fold or at least about two or at least about three foldcompared to the k_(on-r) value of the TNF superfamily trimer binding toits receptor in the absence of the compound and/or the k_(off-r) valueof the compound-trimer complex to the requisite TNF superfamily receptormay be decreased by at least about 1.2-fold, at least about 1.6-fold, atleast about two-fold, more suitably at least about 2.4-fold compared tothe k_(off-r) value of the TNF superfamily trimer binding to itsreceptor in the absence of the compound.

The on-rate for compound binding to TNF superfamily trimer (k_(on-c)) istypically faster than the on-rate for compound-trimer complex binding toTNF superfamily receptor (k_(on-r)). The off-rate for compound-trimercomplex binding to TNF superfamily receptor (k_(off-r)) is alsotypically faster than the off-rate for compound binding to TNFsuperfamily trimer (k_(off-c)). In an embodiment of the invention, theon-rate for compound binding to TNF superfamily trimer (k_(on-c)) isfaster than the on-rate for compound-trimer complex binding to TNFsuperfamily receptor (k_(on-r)), and the off-rate for compound-trimercomplex binding to TNF superfamily receptor (k_(off-r)) is faster thanthe off-rate for compound binding to TNF superfamily trimer (k_(off-c)).The K_(D-c) value of the compound for binding to TNF superfamily trimeris generally lower than the K_(D-r) value of the compound-trimer complexfor binding to TNF superfamily receptor, i.e. the compound has a higheraffinity for the trimer than the compound-trimer complex has for thereceptor.

The k_(on-r), k_(off-r), and K_(D-r) values for both the compound-trimercomplex and the TNF superfamily trimer to the requisite TNF superfamilyreceptor may be determined using any appropriate technique, for examplesurface plasmon resonance, mass spectrometry and isothermal calorimetry.The K_(D-r) value of the TNF superfamily member for binding to itsreceptor in the presence of the test compound may be 1 μM, 100 nM, 10nM, 5 nM, 1 nM, 100 pM, 10 pM or less (down to a lower value of about 1pM). The K_(D-r) value of the TNF superfamily member for binding to itsreceptor in the presence of the test compound (i.e. in a compound-trimercomplex) may be 1 nM or less. The K_(D-r) value of a compound-trimercomplex for binding to the requisite TNF superfamily receptor may beless than 600 pM, less than 500 pM, less than 400 pM, less than 300 pM,less than 200 pM, less than 100 pM or less than 50 pM (again down to alower value of about 1 pM). The K_(D-r) value of a compound-trimercomplex for binding to the requisite TNF superfamily receptor may beless than about 200 pM (to about 1 pM).

Compounds may be identified by an assay which comprises determining theK_(D-r) of the trimeric form of the TNF superfamily member in a sampleof the TNF superfamily member and the compound; comparing the K_(D-r) ofthe trimeric form of the TNF superfamily member in the sample with acontrol sample; and selecting a compound. A control may be a positivecontrol, which is known to increase the binding affinity of the TNFsuperfamily member to its receptor. Here, the test compound may have aK_(D-r) equal to or less than (better than) the positive control.

The compounds stabilise the trimeric form of the TNF superfamily member.Stabilisation is considered to occur if a test compound increases theproportion of trimer compared to the amount of trimer observed for asample containing the TNF superfamily member and the destabilising agentin the absence of the test compound. The test compound may increase theamount of trimer by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%,400% or more compared to the amount of trimer present in a samplecontaining the TNF superfamily member and the destabilising agent in theabsence of the test compound.

The test compound may also increase the amount of trimer compared tothat observed for a sample of the TNF superfamily member in the absenceof both the destabilising agent and the test compound. The test compoundmay increase the amount of trimer by about 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%,200%, 300%, 400% or more compared to the amount of trimer present in asample containing the TNF superfamily member in the absence of both thedestabilising agent and the test compound.

The test compound may increase the amount of the TNF superfamily memberbound to its receptor by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%,300%, 400% or more compared to the amount of the TNF superfamily memberbound to its receptor in a sample containing the TNF superfamily memberin the absence of the test compound.

The test compounds may enhance the stability of the trimeric form of theTNF superfamily member. Enhanced stability of the trimeric form of theTNF superfamily member is considered to occur if a test compoundincreases the thermal transition midpoint (T_(m)) of the trimeric formof the TNF superfamily member compared to the T_(m) of the trimeric formof the TNF superfamily member observed for a sample containing the TNFsuperfamily member and the destabilising agent in the absence of thetest compound. The T_(m) of the trimeric form of the TNF superfamilymember is the temperature at which 50% of the biomolecules are unfolded.The T_(m) of the trimeric form of the TNF superfamily member in thepresence and/or absence of the test compound may be measured using anyappropriate technique known in the art, for example using differentialscanning calorimetry (DSC) or fluorescence probed thermal denaturationassays.

The test compound may increase the T_(m) of the trimeric form of the TNFsuperfamily member by at least 1° C., at least 2° C., at least 5° C., atleast 10° C., at least 15° C., at least 20° C. or more compared to theT_(m) of the trimeric form of the TNF superfamily member in a samplecontaining the TNF superfamily member in the absence of the testcompound. The test compound may increase the T_(m) of the trimeric formof the TNF superfamily member by at least 1° C., by at least 10° C. andby between 10° C. and 20° C.

The compounds may completely or partially inhibit signalling through aTNF receptor when a TNF superfamily member in the form of acompound-trimer complex binds to the receptor. The compound may act toreduce signalling through a TNF superfamily receptor by at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. Alternatively, thecompounds may increase signalling through a TNF receptor when a TNFsuperfamily member in the form of a compound-trimer complex binds to thereceptor. The compound may act to increase signalling through a TNFsuperfamily receptor by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100% or 200%. Any change in the level of signalling maybe measured by any appropriate technique, including but not limited tomeasuring reporter gene activity by alkaline phosphatase or luciferase,NF-κB translocation using machines such as the Cellomics Arrayscan,phosphorylation of downstream effectors, recruitment of signallingmolecules, or cell death.

The compounds may modulate at least one of the downstream effects ofsignalling through a TNF receptor when a TNF superfamily member in theform of a compound-trimer complex binds to the receptor. Such effectsare discussed herein and include TNF superfamily-induced IL-8, IL17A/F,IL2 and VCAM production, TNF superfamily-induced NF-κB activation andneutrophil recruitment. Standard techniques are known in the art formeasuring the downstream effects of TNF superfamily members. Thecompounds may modulate at least 1, 2, 3, 4, 5, 10 or up to all of thedownstream effects of signalling through a TNF receptor.

The activity of the compounds may be quantified using standardterminology, such as IC₅₀ or half maximal effective concentration (EC₅₀)values. IC₅₀ values represent the concentration of a compound that isrequired for 50% inhibition of a specified biological or biochemicalfunction. EC₅₀ values represent the concentration of a compound that isrequired for 50% of its maximal effect. The compounds may have IC₅₀ orEC₅₀ values of 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 nM, 70nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 1 nM, 100 pM or less(down to a lower value of about 10 pM or 1 pM). IC₅₀ and EC₅₀ values maybe measured using any appropriate technique, for example cytokineproduction can be quantified using ELISA. IC₅₀ and EC₅₀ values can thenbe generated using a standard 4-parameter logistic model also known asthe sigmoidal dose response model.

As mentioned above, examples of compounds which are capable of bindingto TNF and modulating signalling are compounds of formulae (1)-(7).

Modulator-TNF Superfamily Member Complexes

An embodiment of the present invention identified that binding of thecompounds described herein to trimeric forms of TNF superfamily membersresults in a conformational change in the TNF superfamily trimer. In afurther embodiment, the TNF superfamily member trimer takes on adeformed or distorted conformation when bound by a compound as disclosedherein.

For example, when compounds (1)-(7) are bound the soluble domain ofhuman TNFα the TNF retains its trimeric structure but the A and Csubunits move away from each other and C rotates to generate a cleftbetween these subunits.

Without being bound by theory, it is believed that, in the absence of acompound, trimeric TNF superfamily members, including trimeric TNFα, arecapable of binding to three separate dimeric TNF superfamily memberreceptors. Each of the dimeric TNF superfamily member receptors iscapable of binding to two separate TNF superfamily trimers. This resultsin the aggregation of multiple TNF superfamily member trimers and TNFsuperfamily member receptor dimers, creating signalling rafts thatinitiate downstream signalling.

When trimeric TNFα is bound to the compound, the conformation of theresulting complex is deformed. Accordingly, without being bound bytheory, it is believed that, in the presence of a compound as disclosedherein, trimeric TNF superfamily members, including trimeric TNFα, areonly capable of binding to two separate dimeric TNF superfamily memberreceptors. The fact that only two, rather than three, separate dimericTNF superfamily member receptors bind to the trimeric TNF superfamilymember reduces or inhibiting the aggregation of multiple TNF superfamilymember trimers and TNF superfamily member receptor dimers. This reducesor inhibits the formation of signalling rafts and so reduces or inhibitsdownstream signalling.

The antibodies of the invention may be used to detect TNFα with adistorted conformation as a result of the binding of a compound asdisclosed herein. Typically the TNFα with a distorted or deformedconformation is trimeric TNFα. However, antibodies of the invention mayalso bind to other forms of the TNFα. For example, antibodies of theinvention may bind to TNFα monomers.

The TNFα is typically trimeric TNFα and may be TNFα_(s) (or trimericTNFα_(s)).

Accordingly, the invention provides a complex comprising a TNFα trimerand a compound that is bound thereto, whereby the compound-trimercomplex binds to the requisite TNFα receptor and modulates thesignalling induced by the trimer through the receptor, wherein saidcomplex binds to an antibody of the invention with an affinity of atleast 1 nM (i.e. 1 nM or less, down to about 1 pM). The TNF superfamilymember is typically TNFα_(s).

Furthermore, the antibody generally binds to the complex with anaffinity that is at least about 100 times lower (the affinity isimproved at least about 100 times), about 200 times lower, relative tothe affinity for binding to the compound in the absence of the TNFαtimer and/or for binding to the TNFα trimer in the absence of compound.

The present invention further provides a compound that is capable ofbinding to a TNFα trimer to form a complex, whereby the compound-trimercomplex binds to the requisite TNFα receptor and modulates thesignalling induced by the trimer through the receptor, wherein thecompound-trimer complex binds to an antibody of the invention with aK_(D-ab) of 1 nM or less (down to about 1 pM). The TNFα is typicallyTNFα_(s).

The antibody may bind to the complex with an affinity that is at leastabout 100 times lower (the affinity is improved at least about 100times), about 200 times lower, relative to the affinity for binding tothe compound in the absence of the TNFα timer and/or for binding to theTNFα trimer in the absence of compound.

The compound-trimer complex may bind to any antibody of the invention.

A compound or complex described herein may be used in the treatmentand/or prophylaxis of a pathological condition. Accordingly, provided isa compound or complex of the invention for use in a method of therapypracticed on the human or animal body. The invention also provides amethod of therapy comprising the administration of a compound or complexof the invention to a subject. The compound or complex of the inventionmay be used in any therapeutic indication and/or pharmaceuticalcomposition described herein.

Antibodies

The invention provides antibodies that selectively bind to at least onecompound-trimer complex comprising at least one compound disclosedherein and a TNFα trimer.

Typically, selective binding of an antibody of the invention to acompound-(trimer) complex is measured relative to the binding of theantibody to the compound in the absence of the TNFα, or to the TNFα inthe absence of the compound or to other (different) compound-(trimer)complexes.

The compound may be any compound described herein, including compounds(1)-(7) (or salts or solvates thereof). A TNF superfamily member isTNFα. Tithe TNFα may be human TNFα, particularly soluble TNFα(TNFα_(s)). The TNFα_(s) may have the sequence of SEQ ID NO: 35 or SEQID NO: 36 (which lacks the N-terminal “S” from SEQ ID NO:35) or may be avariant thereof. Such variants typically retain at least about 60%, 70%,80%, 90%, 91%, 92%, 93%, 94% or 95% identity to SEQ ID NO:35 or SEQ IDNO:36 (or even about 96%, 97%, 98% or 99% identity). In other words,such variants may retain about 60%-about 99% identity to SEQ ID NO:35 orSEQ ID NO:36, about 80%-about 99% identity to SEQ ID NO:35 or SEQ IDNO:36, about 90%-about 99% identity to SEQ ID NO:35 or SEQ ID NO:36 andabout 95%-about 99% identity to SEQ ID NO:35 or SEQ ID NO:36. Variantsare described further below.

Antibodies of the invention may also bind to other forms of TNFα. Toillustrate, the Examples of the present application demonstrate that theCA185_0179 antibody binds to trimeric TNFα. However, as shown in FIG. 1(crystal structure of the CA185_0179 antibody bound to a TNFα monomer inthe presence of compound (1)) the antibody also appears to bind tomonomeric TNFα. Without being bound by theory, in the presence of thecompound it is believed that the soluble domain of the TNF retains itstrimeric structure. However, the A and C subunits move away from eachother (and the C subunit rotates) to generate a cleft between these twosubunits. Thus although antibodies of the invention bind to distortedtrimers, it is also possible that the antibodies can still bind if thetrimeric structure is forced apart into monomers. (See below for furtherdiscussion of the “A” and “C” subunits.)

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An antibody refers to a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen-binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asHCVR or V_(H)) and a heavy chain constant region. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor V_(L)) and a light chain constant region. The variable regions of theheavy and light chains contain a binding domain that interacts with anantigen. The V_(H) and V_(L) regions can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR).

The constant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

An antibody of the invention may be a monoclonal antibody or apolyclonal antibody, and will typically be a monoclonal antibody. Anantibody of the invention may be a chimeric antibody, a CDR-graftedantibody, a nanobody, a human or humanised antibody or anantigen-binding portion of any thereof. For the production of bothmonoclonal and polyclonal antibodies, the experimental animal istypically a non-human mammal such as a goat, rabbit, rat or mouse butthe antibody may also be raised in other species.

Polyclonal antibodies may be produced by routine methods such asimmunisation of a suitable animal, with the antigen of interest. Bloodmay be subsequently removed from the animal and the IgG fractionpurified.

Antibodies generated against compound-trimer complexes of the inventionmay be obtained, where immunisation of an animal is necessary, byadministering the polypeptides to an animal, e.g. a non-human animal,using well-known and routine protocols, see for example Handbook ofExperimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell ScientificPublishers, Oxford, England, 1986). Many warm-blooded animals, such asrabbits, mice, rats, sheep, cows, camels or pigs may be immunized.However, mice, rabbits, pigs and rats are generally most suitable.

Monoclonal antibodies may be prepared by any method known in the artsuch as the hybridoma technique (Kohler & Milstein, 1975, Nature,256:495-497), the trioma technique, the human B-cell hybridoma technique(Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridomatechnique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

Antibodies of the invention may also be generated using singlelymphocyte antibody methods by cloning and expressing immunoglobulinvariable region cDNAs generated from single lymphocytes selected for theproduction of specific antibodies by for example the methods describedby Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15):7843-78481; WO92/02551; WO2004/051268 and WO2004/106377.

The antibodies of the present invention can also be generated usingvarious phage display methods known in the art and include thosedisclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50),Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough etal. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997 1879-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.

Fully human antibodies are those antibodies in which the variableregions and the constant regions (where present) of both the heavy andthe light chains are all of human origin, or substantially identical tosequences of human origin, but not necessarily from the same antibody.Examples of fully human antibodies may include antibodies produced, forexample by the phage display methods described above and antibodiesproduced by mice in which the murine immunoglobulin variable andoptionally the constant region genes have been replaced by their humancounterparts e.g. as described in general terms in EP 0546073, U.S. Pat.Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,EP 0438474 and EP 0463151.

Alternatively, an antibody according to the invention may be produced bya method comprising: immunising a non-human mammal with an immunogencomprising a compound-trimer complex of a trimeric TNFα and a compounddisclosed herein; obtaining an antibody preparation from said mammal;deriving therefrom monoclonal antibodies that selectively recognise saidcomplex and screening the population of monoclonal antibodies formonoclonal antibodies that bind to TNFα only in the presence of thecompound.

The antibody molecules of the present invention may comprise a completeantibody molecule having full length heavy and light chains or afragment or antigen-binding portion thereof. The term “antigen-bindingportion” of an antibody refers to one or more fragments of an antibodythat retain the ability to selectively bind to an antigen. It has beenshown that the antigen-binding function of an antibody can be performedby fragments of a full-length antibody. The antibodies and fragments andantigen binding portions thereof may be, but are not limited to Fab,modified Fab, Fab′, modified Fab′, F(ab′)2, Fv, single domain antibodies(e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies,Bis-scFv, diabodies, triabodies, tetrabodies and epitope-bindingfragments of any of the above (see for example Holliger and Hudson,2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, DrugDesign Reviews—Online 2(3), 209-217). The methods for creating andmanufacturing these antibody fragments are well known in the art (seefor example Verma et al., 1998, Journal of Immunological Methods, 216,165-181). Other antibody fragments for use in the present inventioninclude the Fab and Fab′ fragments described in International patentapplications WO 2005/003169, WO 2005/003170 and WO 2005/003171 andFab-dAb fragments described in International patent applicationWO2009/040562. Multi-valent antibodies may comprise multiplespecificities or may be monospecific (see for example WO 92/22853 and WO05/113605). These antibody fragments may be obtained using conventionaltechniques known to those of skill in the art, and the fragments may bescreened for utility in the same manner as intact antibodies.

The constant region domains of the antibody molecule of the presentinvention, if present, may be selected having regard to the proposedfunction of the antibody molecule, and in particular the effectorfunctions which may be required. For example, the constant regiondomains may be human IgA, IgD, IgE, IgG or IgM domains. In particular,human IgG constant region domains may be used, especially of the IgG1and IgG3 isotypes when the antibody molecule is intended for therapeuticuses and antibody effector functions are required. Alternatively, IgG2and IgG4 isotypes may be used when the antibody molecule is intended fortherapeutic purposes and antibody effector functions are not required.

An antibody of the invention may be prepared, expressed, created orisolated by recombinant means, such as (a) antibodies isolated from ananimal (e.g., a mouse) that is transgenic or transchromosomal for theimmunoglobulin genes of interest or a hybridoma prepared therefrom, (b)antibodies isolated from a host cell transformed to express the antibodyof interest, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of immunoglobulin gene sequences to other DNA sequences.

An antibody of the invention may be a human antibody or a humanisedantibody. The term “human antibody”, as used herein, is intended toinclude antibodies having variable regions in which both the frameworkand CDR regions are derived from human germline immunoglobulinsequences. Furthermore, if the antibody contains a constant region, theconstant region also is derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo). However, the term “human antibody”, asused herein, is not intended to include antibodies in which CDRsequences derived from the germline of another mammalian species, suchas a mouse, have been grafted onto human framework sequences.

Such a human antibody may be a human monoclonal antibody. Such a humanmonoclonal antibody may be produced by a hybridoma that includes a Bcell obtained from a transgenic nonhuman animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell.

Human antibodies may be prepared by in vitro immunisation of humanlymphocytes followed by transformation of the lymphocytes withEpstein-Barr virus.

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to CDR-graftedantibody molecules in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Additional framework region modifications may bemade within the human framework sequences.

As used herein, the term ‘CDR-grafted antibody molecule’ refers to anantibody molecule wherein the heavy and/or light chain contains one ormore CDRs (including, if desired, one or more modified CDRs) from adonor antibody (e.g. a murine or rat monoclonal antibody) grafted into aheavy and/or light chain variable region framework of an acceptorantibody (e.g. a human antibody). For a review, see Vaughan et al,Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather thanthe entire CDR being transferred, only one or more of the specificitydetermining residues from any one of the CDRs described herein above aretransferred to the human antibody framework (see for example, Kashmiriet al., 2005, Methods, 36, 25-34). In one embodiment only thespecificity determining residues from one or more of the CDRs describedherein above are transferred to the human antibody framework. In anotherembodiment only the specificity determining residues from each of theCDRs described herein above are transferred to the human antibodyframework.

When the CDRs or specificity determining residues are grafted, anyappropriate acceptor variable region framework sequence may be usedhaving regard to the class/type of the donor antibody from which theCDRs are derived, including mouse, primate and human framework regions.Suitably, the CDR-grafted antibody according to the present inventionhas a variable domain comprising human acceptor framework regions aswell as one or more of the CDRs or specificity determining residuesdescribed above. Thus, provided in one embodiment is a neutralisingCDR-grafted antibody wherein the variable domain comprises humanacceptor framework regions and non-human donor CDRs.

Examples of human frameworks which can be used in the present inventionare KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). Forexample, KOL and NEWM can be used for the heavy chain, REI can be usedfor the light chain and EU, LAY and POM can be used for both the heavychain and the light chain. Alternatively, human germline sequences maybe used; these are available for example at: http://www.vbase2.org/ (seeRetter et al, Nucl. Acids Res. (2005) 33 (supplement 1), D671-D674).

In a CDR-grafted antibody of the present invention, the acceptor heavyand light chains do not necessarily need to be derived from the sameantibody and may, if desired, comprise composite chains having frameworkregions derived from different chains.

Also, in a CDR-grafted antibody of the present invention, the frameworkregions need not have exactly the same sequence as those of the acceptorantibody. For instance, unusual residues may be changed to morefrequently occurring residues for that acceptor chain class or type.Alternatively, selected residues in the acceptor framework regions maybe changed so that they correspond to the residue found at the sameposition in the donor antibody (see Reichmann et al., 1998, Nature, 332,323-324). Such changes should be kept to the minimum necessary torecover the affinity of the donor antibody. A protocol for selectingresidues in the acceptor framework regions which may need to be changedis set forth in WO 91/09967.

It will also be understood by one skilled in the art that antibodies mayundergo a variety of posttranslational modifications. The type andextent of these modifications often depends on the host cell line usedto express the antibody as well as the culture conditions. Suchmodifications may include variations in glycosylation, methionineoxidation, diketopiperazine formation, aspartate isomerization andasparagine deamidation. A frequent modification is the loss of acarboxy-terminal basic residue (such as lysine or arginine) due to theaction of carboxypeptidases (as described in Harris, R J. Journal ofChromatography 705:129-134, 1995).

In one embodiment the antibody heavy chain comprises a CH1 domain andthe antibody light chain comprises a CL domain, either kappa or lambda.

Biological molecules, such as antibodies or fragments, contain acidicand/or basic functional groups, thereby giving the molecule a netpositive or negative charge. The amount of overall “observed” chargewill depend on the absolute amino acid sequence of the entity, the localenvironment of the charged groups in the 3D structure and theenvironmental conditions of the molecule. The isoelectric point (pI) isthe pH at which a particular molecule or surface carries no netelectrical charge. In one embodiment the antibody or fragment accordingto the present disclosure has an isoelectric point (pI) of at least 7.In one embodiment the antibody or fragment has an isoelectric point ofat least 8, such as 8.5, 8.6, 8.7, 8.8 or 9. In one embodiment the pI ofthe antibody is 8. Programs such as ** ExPASYhttp://www.expasy.ch/tools/pi_tool.html (see Walker, The ProteomicsProtocols Handbook, Humana Press (2005), 571-607) may be used to predictthe isoelectric point of the antibody or fragment.

Antibodies which bind to the epitope disclosed herein may comprise atleast one, at least two or all three heavy chain CDR sequences of SEQ IDNOS: 4 to 6 (HCDR1/HCDR2/HCDR3 respectively). These are theHCDR1/HCDR2/HCDR3 sequences of the CA185_01974 antibody of the Examples.In some instances, the antibody does not comprise CDRs of SEQ ID NOS: 4to 6 (i.e. the antibody does not comprise CDRs of any one of, or all of,SEQ ID NOs: 4-6).

Furthermore, such antibodies may comprise at least one, at least two orall three light chain CDR sequences of SEQ ID NOS: 1 to 3(LCDR1/LCDR2/LCDR3 respectively). These are the LCDR1/LCDR2/LCDR3sequences of the CA185_01974 antibody of the Examples. In someinstances, the antibody does not comprise CDRs of SEQ ID NOs: 1 to 3(i.e. the antibody does not comprise CDRs of any one of, or all of, SEQID NOs: 1 to 3).

The antibody comprises at least a HCDR3 sequence of SEQ ID NO: 6. In oneembodiment, the antibody comprises at least one heavy chain CDR sequenceselected from SEQ ID NOS: 4 to 6 and at least one light chain CDRsequence selected from SEQ ID NOS 1 to 3. The antibody may comprise atleast two heavy chain CDR sequences selected from SEQ ID NOS: 4 to 6 andat least two light chain CDR sequences selected from SEQ ID NOS: 1 to 3.The antibody comprises all three heavy chain CDR sequences of SEQ IDNOS: 4 to 6 (HCDR1/HCDR2/HCDR3 respectively) and all three light chainCDR sequences SEQ ID NOS: 1 to 3 (LCDR1/LCDR2/LCDR3 respectively). Theantibodies may be chimeric, human or humanised antibodies. In someinstances, the antibody does not comprise CDRs of any one of, or all of,SEQ ID NOs: 1 to 6.

The antibody may also comprise at least one, at least two or all threeheavy chain CDR sequences of SEQ ID NOS: 19 to 21 (HCDR1/HCDR2/HCDR3respectively). These are the HCDR1/HCDR2/HCDR3 sequences of theCA185_01979 antibody of the Examples. In some instances, the antibodydoes not comprise CDRs of SEQ ID NOs: 19 to 21 (i.e. CDRs of any one of,or all of, SEQ ID NOs: 19-21).

The antibody comprises a HCDR3 sequence of SEQ ID NO: 21.

The antibody may also comprise at least one, at least two or all threelight chain CDR sequences of SEQ ID NOS: 1, 17, 18 (LCDR1/LCDR2/LCDR3respectively). These are the LCDR1/LCDR2/LCDR3 sequences of theCA185_01979 antibody of the Examples. In some instances, the antibodydoes not comprise CDRs of SEQ ID NOs: 1, 17 or 18 (i.e. CDRs of any oneof, or all of, SEQ ID NOs: 1, 17 or 18.)

In one embodiment of the invention, the antibody comprises at least oneheavy chain CDR sequence selected from SEQ ID NOS: 19 to 21 and at leastone light chain CDR sequence selected from SEQ ID NOS: 1, 17, 18. Theantibody may comprise at least two heavy chain CDR sequences selectedfrom SEQ ID NOS: 19 to 21 and at least two light chain CDR sequencesselected from SEQ ID NOS: 1, 17, 18. The antibody may comprise all threeheavy chain CDR sequences of SEQ ID NOS: 19 to 21 (HCDR1/HCDR2/HCDR3respectively) and all three light chain CDR sequences SEQ ID NOS: 1, 17,18 (LCDR1/LCDR2/LCDR3 respectively). The antibodies may be chimeric,human or humanised antibodies. In some instances, the antibody does notcomprise CDRs of any one of, or all of, SEQ ID NOs: 1, 17, 18, 19, 20 or21.

The antibody may comprise any combination of CDR sequences of theCA185_01974 antibody and the CA185_01979 antibody. In particular, theantibody may comprise least one HCDR sequence selected from SEQ ID NOs:4-6 and 19-21 and/or at least one LCDR sequence selected from SEQ IDNOs: 1-3, 17 and 18.

The antibody may comprise:

-   -   a HCDR1 selected from SEQ ID NOs: 4 and 19; and/or    -   a HCDR2 selected from SEQ ID NOs: 5 and 20; and/or    -   a HCDR3 selected from SEQ ID NOs: 6 and 21; and/or    -   a LCDR1 of SEQ ID NO: 1; and/or    -   a LCDR2 selected from SEQ ID NOs: 2 and 17; and/or    -   a LCDR3 selected from SEQ ID NOs: 3 and 18.

In some instances, the antibody does not comprise CDRs of any one of thefollowing sequences: SEQ ID NOs: 1 to 6 and 17-21.

The antibody may comprise a heavy chain variable region (HCVR) sequenceof SEQ ID NO: 8 (the HCVR of CA185_01974). The antibody may comprise alight chain variable region (LCVR) sequence of SEQ ID NO: 7 (the LCVR ofCA185_01974). The antibody suitably comprises the heavy chain variableregion sequence of SEQ ID NO: 8 and the light chain variable regionsequence of SEQ ID NO: 7.

In some instances, the antibody does not comprise a HCVR of SEQ ID NO: 8and/or an LCVR of SEQ ID NO: 7.

The antibody may also comprise a heavy chain variable region (HCVR)sequence of SEQ ID NO: 23 (the HCVR of CA185_01979). The antibody maycomprise a light chain variable region (LCVR) sequence of SEQ ID NO: 22(the LCVR of CA185_01979). The antibody suitably comprises the heavychain variable region sequence of SEQ ID NO: 23 and the light chainvariable region sequence of SEQ ID NO: 22. In some instances, theantibody does not comprise a HCVR of SEQ ID NO: 23 and/or an LCVR of SEQID NO: 22.

The antibody may comprise a combination of heavy and light chainvariable regions from the CA185_01974 and CA185_01979 antibodies. Inother words, the antibody may comprise a heavy chain variable region ofSEQ ID NO: 8 or 23 and/or a light chain variable region of SEQ ID NO: 7or 22.

In some instances, the antibody does not comprise a heavy chain variableregion of SEQ ID NO: 8 or 23 and/or a light chain variable region of SEQID NO: 7 or 22.

The antibody may comprise a heavy chain (H-chain) sequence of SEQ ID NO:12 (CA185_01974 mIgG1) or 13 (CA185_01974 mFab (no hinge)). The antibodymay comprise a light chain (L-chain) sequence of SEQ ID NO: 11(CA185_01974 kappa light chain). The antibody may comprise the heavychain sequence of SEQ ID NO: 12/13 and the light chain sequence of SEQID NO: 11. The antibodies may be chimeric, human or humanisedantibodies. In some instances, the antibody does not comprise heavyand/or light chains of these sequences.

The antibody may comprise a heavy chain sequence of SEQ ID NO: 27(CA185_01979 mIgG1) or 28 (CA185_01979 mFab (no hinge)). The antibodymay comprise a light chain sequence of SEQ ID NO: 26 (CA185_01979 kappalight chain). The antibody comprises the heavy chain sequence of SEQ IDNO: 27/28 and the light chain sequence of SEQ ID NO: 26. The antibodiesmay be chimeric, human or humanised antibodies. Again, sequences fromCA185_01974 and CA185_01979 may be combined. In some instances, theantibody does not comprise heavy and/or light chains of these sequences.

The antibody may alternatively be or may comprise a variant of one ofthe specific sequences recited above. For example, a variant may be asubstitution, deletion or addition variant of any of the above aminoacid sequences.

A variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up to 20 ormore (typically up to a maximum of 50) amino acid substitutions and/ordeletions from the specific sequences discussed above. “Deletion”variants may comprise the deletion of individual amino acids, deletionof small groups of amino acids such as 2, 3, 4 or 5 amino acids, ordeletion of larger amino acid regions, such as the deletion of specificamino acid domains or other features. “Substitution” variants typicallyinvolve the replacement of one or more amino acids with the same numberof amino acids and making conservative amino acid substitutions. Forexample, an amino acid may be substituted with an alternative amino acidhaving similar properties, for example, another basic amino acid,another acidic amino acid, another neutral amino acid, another chargedamino acid, another hydrophilic amino acid, another hydrophobic aminoacid, another polar amino acid, another aromatic amino acid or anotheraliphatic amino acid. Some properties of the 20 main amino acids whichcan be used to select suitable substituents are as follows:

Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral Cys polar,hydrophobic, neutral Asn polar, hydrophilic, neutral Asp polar,hydrophilic, charged (−) Pro hydrophobic, neutral Glu polar,hydrophilic, charged (−) Gln polar, hydrophilic, neutral Phe aromatic,hydrophobic, neutral Arg polar, hydrophilic, charged (+) Gly aliphatic,neutral Ser polar, hydrophilic, neutral His aromatic, polar,hydrophilic, Thr polar, hydrophilic, neutral charged (+) Ile aliphatic,hydrophobic, neutral Val aliphatic, hydrophobic, neutral Lys polar,hydrophilic, charged (+) Trp aromatic, hydrophobic, neutral Leualiphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic

“Derivatives” or “variants” generally include those in which instead ofthe naturally occurring amino acid the amino acid which appears in thesequence is a structural analog thereof. Amino acids used in thesequences may also be derivatized or modified, e.g. labelled, providingthe function of the antibody is not significantly adversely affected.

Derivatives and variants as described above may be prepared duringsynthesis of the antibody or by post-production modification, or whenthe antibody is in recombinant form using the known techniques ofsite-directed mutagenesis, random mutagenesis, or enzymatic cleavageand/or ligation of nucleic acids.

Variant antibodies may have an amino acid sequence which has more thanabout 60%, or more than about 70%, e.g. 75 or 80%, more than about 85%,e.g. more than about 90 or 95% amino acid identity to the amino acidsequences disclosed herein (particularly the HCVR/LCVR sequences and theH- and L-chain sequences). Furthermore, the antibody may be a variantwhich has more than about 60%, or more than about 70%, e.g. 75 or 80%,more than about 85%, e.g. more than about 90 or 95% amino acid identityto the HCVR/LCVR sequences and the H- and L-chain sequences disclosedherein, whilst retaining the exact CDRs disclosed for these sequences.Variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity to the HCVR/LCVR sequences and to the H- andL-chain sequences disclosed herein (in some circumstances whilstretaining the exact CDRs). In some instances, the antibody is not avariant with more than 60, 70, 75, 80, 85, 90 or 95% identity to any ofthe sequences described herein (i.e. the antibody does not have 60, 70,75, 80, 85, 90 or 95% identity to the HCVR and/or LCVR sequences, or theH- and/or L-chain sequences described herein).

Variants retain about 60%-about 99% identity, about 80%-about 99%identity, about 90%-about 99% identity or about 95%-about 99% identity.This level of amino acid identity may be seen across the full length ofthe relevant SEQ ID NO sequence or over a part of the sequence, such asacross about 20, 30, 50, 75, 100, 150, 200 or more amino acids,depending on the size of the full length polypeptide.

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters—Method: accurate, Matrix: PAM, Gap openpenalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters—Matrix: PAM, Gap open penalty: 10.00, %identity for delay: 30, Penalize end gaps: on, Gap separation distance:0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specificgap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues:GPSNDQEKR. Sequence identity at a particular residue is intended toinclude identical residues which have simply been derivatized.

Antibodies having specific sequences and variants which maintain thefunction or activity of these chains are therefore provided.

The present invention also provides an isolated DNA sequence encodingthe heavy and/or light chain variable regions(s) of an antibody moleculeof the present invention.

An isolated DNA sequence of SEQ ID NO: 10 encodes the heavy chainvariable region of SEQ ID NO: 8. An isolated DNA sequence of SEQ ID NO:9 encodes the light chain variable region of SEQ ID NO: 7.

An isolated DNA sequence of SEQ ID NO: 25 encodes the heavy chainvariable region of SEQ ID NO: 23. An isolated DNA sequence of SEQ ID NO:24 encodes the light chain variable region of SEQ ID NO: 22.

The present invention also provides an isolated DNA sequence encodingthe heavy and/or light chain(s) of any antibody molecule of the presentinvention. Suitably, the DNA sequence encodes the heavy or the lightchain of an antibody molecule of the present invention.

The isolated DNA sequences of SEQ ID NOs: 15 or 16 encode the heavychains of SEQ ID NOs: 12 and 13 respectively. The isolated DNA sequenceof SEQ ID NO: 14 encodes the light chain of SEQ ID NO: 11.

The isolated DNA sequences of SEQ ID NOs: 30 or 31 encode the heavychains of SEQ ID NOs: 27 and 28 respectively. The isolated DNA sequenceof SEQ ID NO: 29 encodes the light chain of SEQ ID NO: 26.

A suitable polynucleotide sequence may alternatively be a variant of oneof these specific polynucleotide sequences. For example, a variant maybe a substitution, deletion or addition variant of any of the abovenucleic acid sequences. A variant polynucleotide may comprise 1, 2, 3,4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or morenucleic acid substitutions and/or deletions from the sequences given inthe sequence listing. Generally, a variant has 1-20, 1-50, 1-75 or 1-100substitutions and/or deletions.

Suitable variants may be at least about 70% homologous to apolynucleotide of any one of nucleic acid sequences disclosed herein,typically at least about 80 or 90% and at least about 95%, 97% or 99%homologous thereto. Variants may retain at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Variants retain about60%-about 99% identity, about 80%-about 99% identity, about 90%-about99% identity or about 95%-about 99% identity. Homology and identity atthese levels is generally present at least with respect to the codingregions of the polynucleotides. Methods of measuring homology are wellknown in the art and it will be understood by those of skill in the artthat in the present context, homology is calculated on the basis ofnucleic acid identity. Such homology may exist over a region of at leastabout 15, at least about 30, for instance at least about 40, 60, 100,200 or more contiguous nucleotides (depending on the length). Suchhomology may exist over the entire length of the unmodifiedpolynucleotide sequence.

Methods of measuring polynucleotide homology or identity are known inthe art. For example the UWGCG Package provides the BESTFIT programwhich can be used to calculate homology (e.g. used on its defaultsettings) (Devereux et al (1984) Nucleic Acids Research 12, p 387-395).

The PILEUP and BLAST algorithms can also be used to calculate homologyor line up sequences (typically on their default settings), for exampleas described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul,S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4,and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90:5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to the second sequenceis less than about 1, typically less than about 0.1, suitably less thanabout 0.01, and most suitably less than about 0.001. For example, thesmallest sum probability may be in the range of about 1-about 0.001,often about 0.01-about 0.001.

The homologue may differ from a sequence in the relevant polynucleotideby less than about 3, 5, 10, 15, 20 or more mutations (each of which maybe a substitution, deletion or insertion). For example, the homologuemay differ by 3-50 mutations, often 3-20 mutations. These mutations maybe measured over a region of at least 30, for instance at least about40, 60 or 100 or more contiguous nucleotides of the homologue.

In one embodiment, a variant sequence may vary from the specificsequences given in the sequence listing by virtue of the redundancy inthe genetic code. The DNA code has 4 primary nucleic acid residues (A,T, C and G) and uses these to “spell” three letter codons whichrepresent the amino acids the proteins encoded in an organism's genes.The linear sequence of codons along the DNA molecule is translated intothe linear sequence of amino acids in the protein(s) encoded by thosegenes. The code is highly degenerate, with 61 codons coding for the 20natural amino acids and 3 codons representing “stop” signals. Thus, mostamino acids are coded for by more than one codon—in fact several arecoded for by four or more different codons. A variant polynucleotide ofthe invention may therefore encode the same polypeptide sequence asanother polynucleotide of the invention, but may have a differentnucleic acid sequence due to the use of different codons to encode thesame amino acids.

The DNA sequence of the present invention may comprise synthetic DNA,for instance produced by chemical processing, cDNA, genomic DNA or anycombination thereof.

DNA sequences which encode an antibody molecule of the present inventioncan be obtained by methods well known to those skilled in the art. Forexample, DNA sequences coding for part or all of the antibody heavy andlight chains may be synthesised as desired from the determined DNAsequences or on the basis of the corresponding amino acid sequences.

General methods by which the vectors may be constructed, transfectionmethods and culture methods are well known to those skilled in the art.In this respect, reference is made to “Current Protocols in MolecularBiology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and theManiatis Manual produced by Cold Spring Harbor Publishing.

Also provided is a host cell comprising one or more cloning orexpression vectors comprising one or more DNA sequences encoding anantibody of the present invention. Any suitable host cell/vector systemmay be used for expression of the DNA sequences encoding the antibodymolecule of the present invention. Bacterial, for example E. coli, andother microbial systems may be used or eukaryotic, for examplemammalian, host cell expression systems may also be used. Suitablemammalian host cells include CHO, myeloma or hybridoma cells.

The present invention also provides a process for the production of anantibody molecule according to the present invention comprisingculturing a host cell containing a vector of the present invention underconditions suitable for leading to expression of protein from DNAencoding the antibody molecule of the present invention, and isolatingthe antibody molecule.

Screening methods as described herein may be used to identify suitableantibodies that are capable of binding to a compound-trimer complex.Thus, the screening methods described herein may be carried out to testantibodies of interest.

Antibodies of the invention can be tested for binding to acompound-trimer complex by, for example, standard ELISA or Westernblotting. An ELISA assay can also be used to screen for hybridomas thatshow positive reactivity with the target protein. The bindingselectivity of an antibody may also be determined by monitoring bindingof the antibody to cells expressing the target protein, for example byflow cytometry. Thus, a screening method of the invention may comprisethe step of identifying an antibody that is capable of binding acompound-trimer complex by carrying out an ELISA or Western blot or byflow cytometry.

Antibodies of the invention selectively (or specifically) recognise aTNFα trimer-compound complex, i.e. an epitope within the compound-trimercomplex. An antibody, or other compound, “selectively binds” or“selectively recognises” a protein when it binds with preferential orhigh affinity to the protein for which it is selective but does notsubstantially bind, or binds with low affinity, to other proteins. Theselectivity of an antibody of the invention for a target acompound-trimer complex may be further studied by determining whether ornot the antibody binds to other related compound-trimer complexes asdiscussed above or whether it discriminates between them.

An antibody may bind specifically (or selectively) to compound-trimercomplexes comprising other trimeric forms of one or more TNF superfamilymembers. For example, an antibody may bind to compound-trimer complexescomprising TNFα, compound-trimer complexes comprising TNFβ andcompound-trimer complexes comprising CD40L. Alternatively, an antibodymay bind specifically (or selectively) to compound-trimer complexescomprising only TNFα, but not to compound-trimer complexes comprisingany other TNF superfamily members. For example, an antibody may bind tocompound-trimer complexes comprising TNFα, but not to compound-trimercomplexes comprising TNFβ or compound-trimer complexes comprising CD40L.An antibody may bind specifically (or selectively) to compound-trimercomplexes comprising up to two, three, four or up to all of the TNFsuperfamily members.

By specific (or selective), it will be understood that the antibodybinds to the compound-trimer complexes of interest with no significantcross-reactivity to any other molecule, which may include test compoundsin the absence of a TNF superfamily trimer or TNF superfamily membertrimers in the absence of a test compound. Cross-reactivity may beassessed by any suitable method described herein. Cross-reactivity of anantibody for a compound-trimer complex with a molecule other than thecompound-trimer complex may be considered significant if the antibodybinds to the other molecule at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95% or 100%as strongly as it binds to the compound-trimer complex of interest. Anantibody that is specific (or selective) for the compound-trimer complexmay bind to another molecule at less than about 90%, 85%, 80%, 75%, 70%,65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% the strength that itbinds to the compound-trimer complex. The antibody may bind to the othermolecule at less than about 20%, less than about 15%, less than about10% or less than about 5%, less than about 2% or less than about 1% thestrength that it binds to the compound-trimer complex. The antibodyspecifically (or selectively) binds to a compound-trimer complexcompared with (i) the trimeric form of the TNFα in the absence of thecompound and/or (ii) the compound in the absence of the TNFα trimer.

The rates at which an antibody binds to a compound-trimer complex isreferred to herein as the “on” rate” k_(on-a)b and the rate at which theantibody dissociates from the compound-trimer complex is referred toherein as the “off” rate or k_(off-ab). As used herein, the symbol“K_(D-ab)” denotes the binding affinity (dissociation constant) of anantibody for a compound-trimer complex. K_(D-ab) is defined ask_(off-ab)/k_(on-ab). Antibodies may have slow “on” rates, which can bemeasured in minutes by mass spectral analysis of the compound-trimercomplex and antibody peak intensities. K_(D-ab) values for an antibodycan be estimated by repeating this measurement at different antibody:compound-trimer complex ratios.

The K_(D-ab) value of the antibody for binding to a compound-trimercomplex may be at least about 1.5 times, 2 times, 3 times, 4 times, 5times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70times, 80 times, 90 times, 100 times, 200 times, 300 times or 400 timeslower, or lower, than the K_(D-a)b value of the antibody for binding tothe trimeric TNF superfamily member in the absence of the compoundand/or the K_(D-ab) value of the antibody for binding to the compound inthe absence of the trimeric TNF superfamily member. The K_(D-ab) valueof the antibody for binding to a compound-trimer complex may bedecreased at least about 10 times, at least about 100 times, at leastabout 200 times, or at least about 300 times the K_(D-ab) value of theTNF superfamily trimer binding to the TNF superfamily receptor in theabsence of the test compound, i.e. the binding affinity of the antibodyfor the compound-trimer complex is typically increased at least about10-fold, suitably at least about 100-fold, more suitably at least about200-fold, most suitably at least about 300-fold compared to the bindingaffinity of the antibody to the trimeric TNF superfamily member in theabsence of the compound and/or the binding affinity of the antibody tothe compound in the absence of the trimeric TNF superfamily member.

The binding affinity may be given in terms of binding affinities(K_(D-ab)) and may be given in any appropriate units, such as μM, nM orpM. The smaller the K_(D-ab) value, the larger the binding affinity ofthe antibody to the compound-trimer complex.

The K_(D-a)b value of the antibody for binding to the compound-trimercomplex may be at least about 1.5 times, 2 times, 3 times, 4 times, 5times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70times, 80 times, 90 times, 100 times lower, or even lower than theK_(D-ab) value of the antibody for binding to the trimeric TNFsuperfamily member in the absence of the compound and/or the K_(D-ab)value of the antibody for binding to the compound in the absence of thetrimeric TNF superfamily member.

The decrease in the K_(D-ab) value of the antibody for binding to thecompound-trimer complex compared to the K_(D-ab) value of the antibodybinding to the trimeric TNF superfamily member in the absence of thecompound and/or the K_(D-ab) value of the antibody for binding to thecompound in the absence of the trimeric TNF superfamily member mayresult from an increase in the on rate (k_(on-a)b) of the antibodybinding to the compound-trimer complex compared to the antibody bindingto the trimeric TNF superfamily member in the absence of the compoundand/or the antibody binding to the compound in the absence of thetrimeric TNF superfamily member; and/or a decrease in the off rate(k_(off-ab)) compared to the antibody binding to the trimeric TNFsuperfamily member in the absence of the compound and/or the antibodybinding to the compound in the absence of the trimeric TNF superfamilymember.

The on rate (k_(on-ab)) of the antibody binding to the compound-trimercomplex is increased compared to the on rate of the antibody binding tothe trimeric TNF superfamily member in the absence of the compoundand/or the antibody binding to the compound in the absence of thetrimeric TNF superfamily member. The off rate (k_(off-ab)) of theantibody binding to the compound-trimer complex is decreased compared tothe off rate of the antibody binding to the trimeric TNF superfamilymember in the absence of the compound and/or the antibody binding to thecompound in the absence of the trimeric TNF superfamily member. The onrate (k_(on-ab)) of the antibody binding to the compound-trimer complexis increased, and the off-rate (k_(off-ab)) of the antibody binding tothe compound-trimer complex is decreased, compared to the antibodybinding to the trimeric TNF superfamily member in the absence of thecompound and/or the antibody binding to the compound in the absence ofthe trimeric TNF superfamily member.

The k_(on-ab) value of the antibody binding to the compound-trimercomplex may be increased by at least about 1.5-fold or at least two-foldand at least about three fold compared to the k_(on-ab) value of theantibody binding to the trimeric TNF superfamily member in the absenceof the compound and/or the antibody binding to the compound in theabsence of the trimeric TNF superfamily member and/or the k_(off-ab)value of the antibody binding to the compound-trimer complex may bedecreased by at least about two-fold, at least about 10-fold, at leastabout 20-fold, at least about 30-fold, at least about 40-fold, at leastabout 50-fold, at least about 60-fold, at least about 70-fold, at leastabout 80-fold more suitably at least about 90-fold compared to thek_(off-ab) value of the antibody binding to the trimeric TNF superfamilymember in the absence of the compound and/or the antibody binding to thecompound in the absence of the trimeric TNF superfamily member.

The k_(on-ab), k_(off-ab), and K_(D-ab) values may be determined usingany appropriate technique, for example surface plasmon resonance, massspectrometry and isothermal calorimetry.

The K_(D-ab) value of the antibody binding to a compound-trimer complexmay be 1 nM, 900 pM, 700 pM, 500 pM, 100 pM, 10 pM or less (typicallydown to about 1 pM). Antibodies of the invention will bind to thecompound-trimer complexes of the invention with high affinity, forexample in the picomolar range. The K_(D-ab) value of the antibodybinding to a compound-trimer complex may be 1 nM or less, 900 pM orless, 700 pM or less, 500 pM or less, 400 pM or less, 300 pM or less,200 pM or less, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM orless, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pMor less, 10 pM or less (again, down to about 1 pM).

Once a suitable antibody has been identified and selected, the aminoacid sequence of the antibody may be identified by methods known in theart. The genes encoding the antibody can be cloned using degenerateprimers. The antibody may be recombinantly produced by routine methods.

Antibodies may compete for binding to TNFα with, or bind to the sameepitope as, those defined above in terms of H-chain/L-chain, HCVR/LCVRor CDR sequences. In particular, an antibody may compete for binding toTNFα with, or bind to the same epitope as, an antibody which comprises aHCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequence combination of SEQ ID NOs:4/5/6/1/2/3 or SEQ ID NOs: 19/20/21/1/17/18. An antibody may compete forbinding to TNFα with, or bind to the same epitope as, an antibody whichcomprises a HCVR and LCVR sequence pair of SEQ ID NOs: 8/7 or SEQ IDNOs: 23/22.

The term “epitope” is a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those residues that directly contribute to the affinity of theinteraction. Epitopes may also be conformational, that is, composed ofnon-linear amino acids. In certain embodiments, epitopes may includedeterminants that are chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl groups, or sulfonylgroups, and, in certain embodiments, may have specific three-dimensionalstructural characteristics, and/or specific charge characteristics.

One can determine whether an antibody binds to the same epitope as, orcompetes for binding with, a reference antibody by using routine methodsknown in the art. For example, to determine if a test antibody binds tothe same epitope as a reference antibody of the invention, the referenceantibody is allowed to bind to a protein or peptide under saturatingconditions. Next, the ability of a test antibody to bind to the proteinor peptide is assessed. If the test antibody is able to bind to theprotein or peptide following saturation binding with the referenceantibody, it can be concluded that the test antibody binds to adifferent epitope than the reference antibody. On the other hand, if thetest antibody is not able to bind to protein or peptide followingsaturation binding with the reference antibody, then the test antibodymay bind to the same epitope as the epitope bound by the referenceantibody of the invention.

To determine if an antibody competes for binding with a referenceantibody, the above-described binding methodology is performed in twoorientations. In a first orientation, the reference antibody is allowedto bind to a protein/peptide under saturating conditions followed byassessment of binding of the test antibody to the protein/peptidemolecule. In a second orientation, the test antibody is allowed to bindto the protein/peptide under saturating conditions followed byassessment of binding of the reference antibody to the protein/peptide.If, in both orientations, only the first (saturating) antibody iscapable of binding to the protein/peptide, then it is concluded that thetest antibody and the reference antibody compete for binding to theprotein/peptide. As will be appreciated by the skilled person, anantibody that competes for binding with a reference antibody may notnecessarily bind to the identical epitope as the reference antibody, butmay sterically block binding of the reference antibody by binding anoverlapping or adjacent epitope.

Two antibodies bind to the same or overlapping epitope if eachcompetitively inhibits (blocks) binding of the other to the antigen.That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibitsbinding of the other by at least 50%, 75%, 90% or even 99% as measuredin a competitive binding assay (see, e.g., Junghans et al., Cancer Res,1990:50:1495-1502). Alternatively, two antibodies have the same epitopeif essentially all amino acid mutations in the antigen that reduce oreliminate binding of one antibody reduce or eliminate binding of theother. Two antibodies have overlapping epitopes if some amino acidmutations that reduce or eliminate binding of one antibody reduce oreliminate binding of the other.

Additional routine experimentation (e.g., peptide mutation and bindinganalyses) can then be carried out to confirm whether the observed lackof binding of the test antibody is in fact due to binding to the sameepitope as the reference antibody or if steric blocking (or anotherphenomenon) is responsible for the lack of observed binding. Experimentsof this sort can be performed using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art.

The antibodies of the invention bind to an epitope which comprises atleast the following residues of trimeric TNFα: (a) Y115; (b) Q149; and(c) N137 and/or K98 (i.e. N137 or K98 or both N137 and K98), whereinY115 and Q149 are present on the C chain of trimeric TNFα, and N137 andK98 are present on the A chain of trimeric TNFα, and wherein the residuenumbering corresponds to (is according to) TNFα of SEQ ID NO: 36. Asdiscussed further below, SEQ ID NO: 36 represents the sequence of humansoluble TNFα. In some instances, the antibody binds an epitopecomprising all of Y115, Q149, N137 and K98.

Although these residues are provided for a particular sequence of humansoluble TNFα, the skilled person could readily extrapolate the positionsof these residues to other TNFα sequences (mouse or rat) using routinetechniques. Antibodies binding to epitopes comprising the correspondingresidues within these other TNF sequences are therefore also provided bythe invention.

Y115 and Q149 are hidden (and non-accessible) within the TNFα trimer inthe absence of compound. However, when the TNF trimer is bound by acompound disclosed herein, these residues become accessible within thedistorted conformation of the trimer. Accordingly, antibodies of theinvention which bind an epitope comprising at least Y115 and Q149 willrecognise the distorted conformation of the trimer (which modulates TNFsignalling), but not the normal conformation of the trimer in theabsence of compound. K98 is also buried to an extent in the symmetricalTNF trimer.

The “A” and “C” chains refer to the first and third monomers, which makeup the TNF trimer. The antibodies of the invention therefore recognise aconformational epitope spanning two monomers of the TNF trimer.

In more detail, when looking at a crystal structure of a TNFα trimerfrom the side it is approximately shaped like a pyramid/cone. When youlook down the trimer axis with the N- and C-termini of the monomer endspointing towards you then you are looking at the “fat” end of thetrimer. In the distorted structure with compound, a cleft opens betweenA and C subunits in which, without being bound by theory, theantibody-of the invention binds.

Which chain is A, B or C may be ascertained by measuring three distancesbetween three C-alpha atoms of three identical residues—e.g. P117 ineach chain (G121 is also appropriate).

The three distances form a triangle which is equilateral in apo TNF butdistorted when compound is bound. The shortest distance is between BCand the longest between AC (for instance AC=13.8 Å, AB=12.3 Å, BC=10.2Å); thus looking down through the axis of the molecule with N/C terminipointing towards you the longest distance defines C then A chains goinganti-clockwise, then B and C again continuing anti-clockwise.

The antibodies of the invention may bind to an epitope whichadditionally comprises E146 on the C chain of trimeric TNFα, P117 on theC chain of trimeric TNFα, or both E146 and P117. P117 is alsosubstantially buried in the apo form of the TNF trimer (in the absenceof compound). In some instances, an antibody of the invention binds anepitope comprising both E146 and P117.

The antibodies of the invention may also bind to an epitope furthercomprising A145, which residue present on the C chain of trimeric TNFα.

The antibodies of the invention may also bind to an epitope furthercomprising Q88, which residue is present on the A chain of TNFα.

The epitope may also further comprise one or more residues selected fromthe group consisting of:

(a) L63;

(b) L94;

(c) S95; and

(d) A96;

wherein L63 is present on the C chain of trimeric TNFα and L94, S95 andA96 are present on the A chain of trimeric TNFα. These residues are allburied to some extent in the apo form of the TNF trimer.

In some instances, antibodies of the invention bind an epitope furthercomprising all of L63, L94, S95 and A96.

The epitope may also further comprise one or more residues selected fromthe group consisting of:

(a) I97;

(b) L93; and

(c) S81,

wherein I97, L93 and S81 are all present on the A chain of trimericTNFα. In some instances, the antibody binds to an epitope furthercomprising all of these residues.

In addition, the antibody may bind to an epitope further comprising oneor more residues selected from the following group:

(a) K65;

(b) Q67;

(c) P113;

(d) D143;

(e) T77;

(f) T79;

(g) I83;

(h) T89;

(g) K90;

(i) V91;

(j) N92;

(k) E135;

(l) I136; and

(m) R138;

wherein K65, Q67, P113 and D143 are present on the C chain of trimericTNFα and T77, T79, I83, T89, K90, V91, N92, E135, I136 and R138 arepresent on the A chain of trimeric TNFα. In some instances, an antibodyof the invention binds to an epitope further comprising all of theseresidues.

An antibody of the invention, may also bind to an epitope furthercomprising one or more residues selected from the following group:

(a) Y119;

(b) Q47; and

(c) S133,

wherein all of Y119, Q47 and S133 are present on the A chain of trimericTNFα. In some instances, an antibody of the invention may bind anepitope further comprising all of these residues.

In some instances, an antibody of the invention may bind an epitopewhich further comprises either Q27 or R131, or both Q27 and R131,wherein these residues are present on the A chain of trimeric TNFα.

As discussed above, the residue numbering is presented according to SEQID NO: 36.

An antibody of the invention may bind to an epitope comprising all ofthe following residues of TNFα of SEQ ID NO: 36:

(a) T77, T79, T89, K90, V91, N92, L93, L94, S95, A96, I97, E135, I136,N137 and R138, which residues are present on the A chain of trimericTNFα and K65, Q67, Y115, A145, E146 and Q149, which residues are presenton the C chain of trimeric TNFα;

(b) T77, T79, I83, T89, K90, V91, N92, L93, L94, S95, A96, I97, K98,E135, I136, N137 and R138, which residues are present on the A chain oftrimeric TNFα and L63, K65, Q67, P113, Y115, D143, A145, E146 and Q149,which residues are present on the C chain of trimeric TNFα;

(c) T77, T79, S81, I83, Q88, T89, K90, V91, N92, L93, L94, S95, A96,I97, K98, E135, I136, N137 and R138, which residues are present on the Achain of trimeric TNFα and L63, K65, Q67, P113, Y115, P117, D143, A145,E146 and Q149, which residues are present on the C chain of trimericTNFα;

(d) Q27, Q47, T77, T79, S81, I83, Q88, T89, K90, V91, N92, L93, L94,S95, A96, I97, K98, Y119, R131, S133, E135, I136, N137 and R138, whichresidues are present on the A chain of trimeric TNFα and L63, K65, Q67,P113, Y115, P117, D143, A145, E146 and Q149, which residues are presenton the C chain of trimeric TNFα.

To screen for antibodies that bind to a particular epitope, a routinecross-blocking assay such as that described in Antibodies, Harlow andLane (Cold Spring Harbor Press, Cold Spring Harb., N.Y.) can beperformed. Other methods include alanine scanning mutants, peptide blots(Reineke (2004) Methods Mol Biol 248:443-63), or peptide cleavageanalysis. In addition, methods such as epitope excision, epitopeextraction and chemical modification of antigens can be employed (Tomer(2000) Protein Science 9: 487-496). Such methods are well known in theart.

Antibody epitopes may also be determined by x-ray crystallographyanalysis. Antibodies of the present invention may therefore be assessedthrough x-ray crystallography analysis of the antibody bound to the TNFαtrimer. Epitopes may, in particular, be identified in this way bydetermining residues on TNFα within 4 Å of an antibody paratope residue.A 5 Å limit may also be used to determine residues.

Antibodies of the invention may also compete with antibodies which bindan epitope comprising the TNFα residues described herein. Methods ofidentifying such antibodies are discussed in the present invention.

The antibodies of the invention may be used to identify compounds of theinvention as described herein. The antibodies of the invention may alsobe used as target engagement biomarkers. A target engagement biomarkercan be used to detect the engagement, i.e. the binding of a ligand to atarget of interest. In the present case, the antibodies of the inventiononly bind to the complexes of compounds of the invention with trimericTNFα. Therefore, if an antibody of the invention is able to bind to acompound-trimer complex, this is evidence that the ligand (compound) hasbound to the target of interest (TNFα trimer). Antibodies of theinvention can be modified to add a detectable marker as describedherein. Therefore, engagement of a compound of the invention with atarget TNFα may be detected using such an antibody.

The use of antibodies of the invention as target engagement biomarkersis potentially useful in a clinical or pre-clinical environment, where asample may be taken from a subject being treated according to thepresent invention. The sample obtained from the subject may be treatedwith an antibody of the invention in order to determine whether thecompound used to treat the subject has bound to the target TNFα. Thesample obtained from the subject may be any appropriate tissue or fluid,such as blood, plasma or urine. The subject may be any mammal includinghuman.

Accordingly, the invention provides the use of an antibody of theinvention as a target engagement biomarker for the detection of acompound-trimer complex comprising trimeric TNFα and a compound that iscapable of binding to trimeric TNFα, whereby the compound-trimer complexbinds to the requisite TNFα receptor and modulates the signallinginduced by the trimer through the receptor in a sample obtained from asubject. The modulation is suitably antagonism of TNFR1 signalling.

Similarly, the present invention provides a method of detecting targetengagement of a compound to trimeric TNFα, whereby the compound-trimercomplex binds to the requisite receptor and modulates the signallinginduced by the trimer through the receptor, said method comprising:

-   -   (a) obtaining a sample from a subject administered said        compound;    -   (b) contacting an antibody of the invention to said sample and a        control sample, wherein said antibody is detectable;    -   (c) determining the amount of binding of said detectable        antibody to said sample and said control sample,        wherein binding of said detectable antibody to said sample        greater than binding of said detectable antibody to said control        sample indicates target engagement of said compound to said        trimeric TNFα.

Methods of detecting antibodies, and measuring the amount of binding ofan antibody to a target, are well known in the art. Typically,antibodies can be labelled. Such labels include enzymes,biotin/streptavidin, fluorescent proteins and fluorescent dyes.

Binding of an antibody to a target may be measured, for example, by animmunoassay method. Immunoassays include Western Blotting, ELISA,immunofluorescence, immunohistochemistry and flow cytometry. Anyappropriate technique may be used to measure binding of the antibody tothe TNFα.

Binding of the detectable antibody to the sample from a subject who hasbeen administered the compound is compared with binding of the antibodyto a control sample. The control sample may be any appropriate sample.The control sample is typically a “negative control” which isrepresentative of binding of the antibody to the trimeric TNFα in theabsence of the compound. For example, the sample may be obtained fromthe patient prior to administration of the compound. The control mayalso be based on previously determined measurements e.g. from a numberof samples from different subjects in the absence of compound.Measurements from about 5, 10, 20, 50 or 100 subjects may be used indetermining the control value. The control may be an average value, or arange of all the values obtained.

The experimental conditions e.g. methods of detection are the same forthe sample from a subject administered the compound, and for the controlsample. The antibody is also the same in both cases.

Greater binding (increased binding) of the detectable antibody to thesample from the patient administered the compound compared with bindingof the antibody to the control sample is indicative of target engagementof the compound to the trimeric TNFα. In other words, equivalent orlower binding (decreased binding) for the sample from the patientadministered the compound relative to the control indicates that thereis no target engagement of said compound. In other words, no significantdifference in the two amounts indicates that there is no targetengagement.

The skilled person can readily determine when there is increased bindingrelative to the control. For example when the control is a range ofdata, target engagement may be determined based on the spread of thedata, the difference between the control data and the detected bindingof the antibody in the sample in question, and calculated confidencelevels. It is also possible to identify target engagement when thedetected binding for the sample in question is higher than the maximumamount of binding detected in any negative control.

Target engagement may be detected if binding of the antibody isincreased by about 30% or more relative to the highest amount in thecontrol range. Target engagement may also be detected if binding of theantibody is increased by about 40% or more, or about 50% or morerelative to the control range. The same applies when the control is anaverage value, or a single value based on a sample from the patientprior to administration of the compound. There is of course no upperlimit to the percentage increase relative to the control.

An antibody of the invention may be used to screen for a compound thatelicits a conformational change in trimeric TNFα, wherein saidconformational change modulates the signalling of the requisite TNFαreceptor on binding of the trimeric TNFα. In a non-limiting example, themodulation may be antagonism of TNFR1 signalling.

The antibodies of the present invention may be used in the treatmentand/or prophylaxis of a pathological condition. Accordingly, provided isan antibody of the invention for use in a method of therapy practiced onthe human or animal body. The invention also provides a method oftherapy comprising the administration of an antibody of the invention toa subject. The antibody of the invention may be used in any therapeuticindication and/or pharmaceutical composition described herein.

Antibody Assays

As described herein, the present invention provides antibodies thatselectively bind to at least one compound-trimer complex describedherein relative to their binding to the compound alone or to the TNFα inthe absence of the compound. These antibodies may be used to identifyfurther compounds or classes of compounds having the same properties.

Monoclonal antibodies may be generated against TNFα using the standardtechniques described herein. These anti-TNFα antibodies can then bescreened for antibodies that bind to compound-trimer complexes of theinvention, or for monoclonal antibodies for which binding to the TNFα isinhibited by compounds as described herein.

Alternatively, monoclonal antibodies can be generated against particularTNFα trimer-compound complexes. These antibodies can then be screenedfor monoclonal antibodies that selectively bind to the TNFα in thepresence of the compound relative to their binding to the TNFα in theabsence of the compound.

Once an antibody that selectively binds to at least one compound-trimercomplex of the invention relative to its binding to the compound aloneor to the TNFα in the absence of the compound has been generated, it canbe used to screen for other compounds possessing the same activity asthe test compounds. For example, compounds (1)-(7) as described hereincan be used to screen for other compounds.

Accordingly, the invention provides an assay for identifying a compoundof the invention comprising the steps of:

a) performing a binding assay to measure the binding affinity of a testcompound-trimer complex to an antibody of the invention;

b) comparing the binding affinity as measured in step (a) with thebinding affinity of a different compound-trimer complex known to bindwith high affinity to the antibody referred to in step (a); and

c) selecting the compound present in the compound-trimer complex of step(a) if its measured binding affinity is acceptable when considered inthe light of the comparison referred to in step (b).

As will be appreciated, the “different” compound-trimer complex referredto in step (b) above will generally be a complex containing the sameTNFα trimer as the compound-trimer complex of step (a), but a differentcompound. The compound of step (b) may be any of compounds (1)-(7).

By “acceptable” in step (c) is meant that the binding affinity of thecompound-trimer complex referred to in step (a) and the binding affinityof the different compound-trimer complex referred to in step (b) are atleast comparable. The binding affinity of the compound-trimer complexreferred to in step (a) may be at least equal to the binding affinity ofthe different compound-trimer complex referred to in step (b). Thebinding affinity may be even higher (i.e. a lower K_(D) value) for thecompound in step (a) compared with the compound in step (b). Selectivebinding of said antibody to said complex is measured relative to thebinding of said antibody to the TNFα in the absence of the compound orto the compound in the absence of the TNFα.

The binding affinity of the compound-trimer complex referred to in step(a) will generally be superior to the binding affinity of the differentcompound-trimer complex referred to in step (b). The difference in thebinding affinity of the compound-trimer complex referred to in step (a)relative to the binding affinity of the different compound-trimercomplex referred to in step (b) will be within limits of 10-fold,20-fold, 50-fold, 100-fold, 200-fold or 500-fold.

Libraries of compounds can be assayed using the antibodies of theinvention. The library compounds can be incubated with said antibody inthe presence and absence of TNFα. A compound that forms part of acompound-trimer complex that binds to an antibody of the invention onlyin the presence of both the TNFα and the compound is a likely candidateto have the same activity as the compounds described herein. The assaysdisclosed herein may then be used to verify whether the test compound isa compound as described herein.

One or more of the antibodies of the invention may be used in the assay.A generic antibody that is capable of binding to complexes of anycompound of the invention with a particular TNFα may be used in theantibody assay of the invention.

A panel of multiple antibodies of the present invention that arespecific for different compound-trimer complexes may be used in theantibody assay of the invention. The panel of antibodies may include atleast 5, at least 10, at least 15, at least 20, at least 30, at least 40or at least 50 antibodies (for example up to 75 antibodies).

The antibody assay of the present invention may be a high throughputassay that is capable of screening a large number of test compounds overa short space of time to identify compounds of the present invention.

The TNFα and its receptors may be purified or present in mixtures, suchas in cultured cells, tissue samples, body fluids or culture medium.Assays may be developed that are qualitative or quantitative, with thelatter being useful for determining the binding parameters (affinityconstants and kinetics) of the test compound to trimeric forms of TNFα,and also of the binding parameters of the compound-trimer complex to therequisite TNF receptor.

The sample comprising the TNFα and the compound may further comprise adestabilising agent. Destabilising agents, also known as chaotropes,include low molar concentrations (e.g. 1M) of urea, guanidine oracetonitrile, high concentrations (e.g. 6M or higher) of these reagentswill result in complete dissociation of the TNFα trimer and unfolding ofthe constituent TNFα monomeric subunits. The destabilising agent may beDMSO, typically at a concentration of 5%, 10% or higher.

The test compounds may have any or/all of the properties discussedabove.

TNF Superfamily and their Receptors

There are 22 TNF superfamily members currently known: TNFα (TNFSF1A),TNFβ (TNFSF1B), CD40L (TNFSF5), BAFF (TNFSF13B/BlyS), APRIL (TNFSF13),OX40L (TNFSF4), RANKL (TNFSF11/TRANCE), TWEAK (TNFSF12), TRAIL(TNFSF10), TL1A (TNFSF15), LIGHT (TNFSF14), Lymphotoxin, Lymphotoxin β(TNFSF3), 4-1BBL (TNFSF9), CD27L (TNFSF7), CD30L (TNFSF8), EDA(Ectodysplasin), EDA-A1 (Ectodysplasin A1), EDA-A2 (Ectodysplasin A2),FASL (TNFSF6), NGF and GITRL (TNFSF18).

In the invention, the TNF superfamily member is TNFα. TNFα exists inboth a soluble (TNFα_(s)) and membrane-bound form (TNFα_(m)). When TNFαreferred to herein encompasses both the TNFα_(s) and TNFα_(m) forms.TNFα is typically in the TNFα_(s) form. The TNFα_(s) may comprise thesequence of SEQ ID NO: 35 or SEQ ID NO: 36, or a variant thereof (asdescribed above).

The assays of the invention may be used to identify modulators of TNFα.Specifically, the assays of the invention may be used to identifycompounds that bind to TNFα, particularly to trimeric forms of TNFα, andthat stabilise these trimers in a conformation that is capable ofbinding to the requisite TNF receptor, and which modulate signallingthrough said receptor. The TNFα may be TNFα_(s).

The compound described herein may be a modulator of at least TNFα. TheTNFα is typically TNFα_(s).

The compound-trimer complex of the invention includes the trimeric formof TNFα. The TNFα is typically TNFα_(s).

Members of the TNF superfamily bind to, and initiate signalling throughTNF receptors. There are currently 34 known TNF receptors: 4-1BB(TNFRSF9/CD137), NGF R (TNFRSF16), BAFF R (TNFRSF13C), Osteoprotegerin(TNFRSF11B), BCMA (TNFRSF17), OX40 (TNFRSF4), CD27 (TNFRSF7), RANK(TNFRSF11A), CD30 (TNFRSF8), RELT (TNFRSF19L), CD40 (TNFRSF5), TACI(TNFRSF13B), DcR3 (TNFRSF6B), TNFRH3 (TNFRSF26), DcTRAIL R1 (TNFRSF23),DcTRAIL R2 (TNFRSF22), TNF-R1 (TNFRSF1A), TNF-R2 (TNFRSF1B), DR3(TNFRSF25), TRAIL R1 (TNFRSF10A), DR6 (TNFRSF21), TRAIL R2 (TNFRSF10B),EDAR, TRAIL R3 (TNFRSF10C), Fas (TNFRSF6/CD95), TRAIL R4 (TNFRSF10D),GITR (TNFRSF18), TROY (TNFRSF19), HVEM (TNFRSF14), TWEAK R (TNFRSF12A),TRAMP (TNFRSF25), Lymphotoxin β R (TNFRSF3) and XEDAR.

When TNF-R is referred to herein this encompasses both TNF-R1 andTNF-R2, including the extracellular domain (ECD) of TNF-R1 and TNF-R2.The assays of the invention may be used to identify compounds thatmodulate the signalling of TNFα through any requisite TNF receptor. Theassays of the invention may be used to identify compounds that modulatethe signalling of TNFα through TNF-R1 or TNF-R2. The TNF superfamilymember may be TNFα and the TNF receptor may be TNF-R1. In an embodimentof the invention, the TNF superfamily member may be TNFα, and the TNFreceptor may be TNF-R1. The assays of the invention may be used toidentify compounds which act by specifically modulating the signallingof TNFα through TNF-R1. In particular, the compounds may act bymodulating the signalling of TNFα through TNF-R1, but have no effect onsignalling of TNFα through TNF-R2.

Therapeutic Indications

TNFα is the archetypal member of the TNF superfamily. TNFα is apleiotropic cytokine that mediates immune regulation and inflammatoryresponses. In vivo, TNFα is also known to be involved in responses tobacterial, parasitic and viral infections. In particular, TNFα is knownto have a role in rheumatoid arthritis (RA), inflammatory bowel diseases(including Crohn's disease), psoriasis, Alzheimer's disease (AD),Parkinson's disease (PD), pain, epilepsy, osteoporosis, asthma, sepsis,fever, Systemic lupus erythematosus (SLE) and Multiple Sclerosis (MS)and cancer. TNFα is also known to have a role in Amyotrophic LateralSclerosis (ALS), ischemic stroke, immune complex-mediatedglomerulonephritis, lupus nephritis (LN), antineutrophil cytoplasmicantibodies (ANCA-) associated glomerulonephritis, minimal changedisease, diabetic nephropathy (DN), acute kidney injury (AKI),obstructive uropathy, kidney allograft rejection, cisplatin-induced AKIand obstructive uropathy.

Other members of the TNF superfamily are known to be involved inautoimmune disease and immune deficiencies. In particular, members ofthe TNF superfamily are known to be involved in RA, SLE, cancer, MS,asthma, rhinitis, osteoporosis and multiple myeloma (MM). TL1A is knownto play a role in organ transplant rejection.

A compound described herein may be used to treat, prevent or ameliorateany condition that can be treated, prevented or ameliorated by aconventional TNF superfamily member modulator. The compound may be usedalone or in combination with a conventional TNF superfamily membermodulator. Any condition that results, partially or wholly, frompathogenic signalling through a TNF receptor by a TNF superfamily memberor from a deficiency in signalling through a TNF receptor by a TNFsuperfamily member may in principle be treated, prevented orameliorated. Pathogenic signalling through a TNF receptor by a TNFsuperfamily member includes but is not limited to increased signallingthrough a TNF receptor over and above the normal physiological level ofsignalling, signalling through a TNF receptor which is initiatednormally, but which fails to stop in response to normal physiologicalsignals and signalling through a TNF receptor that is within the normalphysiological range of magnitude, but which is initiated bynon-physiological means. The invention relates to the treatment,prevention or amelioration of conditions mediated or influenced by TNFα.

The compounds that interact with TNFα are accordingly beneficial in thetreatment and/or prevention of various human ailments. These includeautoimmune and inflammatory disorders; neurological andneurodegenerative disorders; pain and nociceptive disorders; andcardiovascular disorders.

Inflammatory and autoimmune disorders include systemic autoimmunedisorders, autoimmune endocrine disorders and organ-specific autoimmunedisorders. Systemic autoimmune disorders include systemic lupuserythematosus (SLE), psoriasis, vasculitis, polymyositis, scleroderma,multiple sclerosis, ankylosing spondylitis, rheumatoid arthritis andSjögren's syndrome. Autoimmune endocrine disorders include thyroiditis.Organ-specific autoimmune disorders include Addison's disease,haemolytic or pernicious anaemia, glomerulonephritis (includingGoodpasture's syndrome), Graves' disease, idiopathic thrombocytopenicpurpura, insulin-dependent diabetes mellitus, juvenile diabetes,uveitis, inflammatory bowel disease (including Crohn's disease andulcerative colitis), pemphigus, atopic dermatitis, autoimmune hepatitis,primary biliary cirrhosis, autoimmune pneumonitis, autoimmune carditis,myasthenia gravis, spontaneous infertility, osteoporosis, asthma andmuscular dystrophy (including Duchenne muscular dystrophy).

Neurological and neurodegenerative disorders include Alzheimer'sdisease, Parkinson's disease, Huntington's disease, stroke, amyotrophiclateral sclerosis, spinal cord injury, head trauma, seizures andepilepsy.

Cardiovascular disorders include thrombosis, cardiac hypertrophy,hypertension, irregular contractility of the heart (e.g. during heartfailure), and sexual disorders (including erectile dysfunction andfemale sexual dysfunction).

A compound may be used to treat or prevent inflammatory disorders, CNSdisorders, immune disorders and autoimmune diseases, pain, osteoporosis,fever and organ transplant rejection. A compound may be used to treat orprevent rheumatoid arthritis, inflammatory bowel diseases (includingCrohn's disease), psoriasis, Alzheimer's disease, Parkinson's disease,epilepsy, asthma, sepsis, systemic lupus erythematosus, multiplesclerosis, asthma, rhinitis, cancer and osteoporosis. A compound may beused to treat or prevent rheumatoid arthritis (RA), non specificinflammatory arthritis, erosive bone disease, chondritis, cartilagedegeneration and/or destruction, juvenile inflammatory arthritis,Still's Disease (juvenile and/or adult onset), juvenile idiopathicarthritis, juvenile idiopathic arthritis (both oligoarticular andpolyarticular forms), inflammatory bowel diseases (including Crohn'sdisease, ulcerative colitis, indeterminate colitis, pouchitis),psoriasis, psoriatic arthopathy, ankylosing spondylitis, Sjogren'sDisease, Alzheimer's disease (AD), Behcet's Disease, Parkinson's disease(PD), amyotrophic lateral sclerosis (ALS), ischemic stroke, pain,epilepsy, osteoporosis, osteopenia, anaemia of chronic disease,cachexia, diabetes, dyslipidemia, metabolic syndrome, asthma, chronicobstructive airways (or pulmonary) disease, sepsis, fever, respiratorydistress syndrome, systemic lupus erythematosus (SLE), multiplesclerosis (MS) immune complex-mediated glomerulonephritis, lupusnephritis (LN), antineutrophil cytoplasmic antibodies (ANCA-) associatedglomerulonephritis, minimal change disease, diabetic nephropathy (DN),acute kidney injury (AKI), obstructive uropathy, kidney allograftrejection, cisplatin-induced AKI and obstructive uropathy, eye diseases(including diabetic retinopathy, diabetic macular oedema, retinopathy ofprematurity, age related macular degeneration, macular oedema,proliferative and/or non proliferative retinopathy, cornealvascularisation including neovascularization, retinal vein occlusion,various forms of uveitis and keratitis), thyroiditis, fibrosingdisorders including various forms of hepatic fibrosis, various forms ofpulmonary fibrosis, systemic sclerosis, scleroderma, cancer and cancerassociated complications (including skeletal complications, cachexia andanaemia).

As discussed above, antibodies of the present invention may be used astarget engagement biomarkers to assess the effectiveness of treatmentwith a compound or complex as described herein. In one embodiment, asample taken from a subject treated with a compound or complex describedherein may be contacted with an antibody of the invention. The antibodymay then be used to determine the amount of TNFα-compound complexpresent within the sample. The amount of complex determined using theantibody may be related to the effectiveness of the treatment. Forexample, the more complex detected by the antibody of the invention, themore effective the treatment. The amount of complex determined using theantibody is directly proportional to the effectiveness of the treatment.For example, doubling the amount of complex determined using theantibody may be indicative of a doubling of the effectiveness of thetreatment.

An antibody of the invention may be used to determine the amount ofcompound-trimer complex using any appropriate technique. Standardtechniques are known in the art and are disclosed herein. For example,ELISA and Western blotting with an antibody of the invention may be usedto determine the amount of compound-trimer complex.

The amount of the compound-trimer complex may be determined by measuringthe mass of the compound-trimer complex, the concentration of thecompound-trimer complex, and the molarity of the compound-trimercomplex. This amount may be given in any appropriate units. For example,the concentration of the compound-trimer complex may be given in pg/ml,ng/ml or μg/ml. The mass of the compound-trimer complex may be given inpg, ng or μg.

The amount of the compound-trimer complex in a sample of interest may becompared with the level of the compound-trimer complex in anothersample, such as a control sample, as described herein. In such a method,the actual amount of the compound-trimer complex, such as the mass,molar amount, concentration or molarity of the compound-trimer complexin the samples may be assessed. The amount of the compound-trimercomplex may be compared with that in another sample without quantifyingthe mass, molar amount, concentration or molarity of the compound-trimercomplex. Thus, the amount of the compound-trimer complex in a sampleaccording to the invention may be assessed as a relative amount, such asa relative mass, relative molar amount, relative concentration orrelative molarity of the compound-trimer complex based on a comparisonbetween two or more samples.

Pharmaceutical Compositions, Dosages and Dosage Regimes

An antibody, compound or complex of the invention may be provided in apharmaceutical composition. The pharmaceutical composition that willnormally be sterile and will typically include a pharmaceuticallyacceptable carrier and/or adjuvant. A pharmaceutical composition of thepresent invention may additionally comprise a pharmaceuticallyacceptable adjuvant and/or carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier may be suitable for parenteral,e.g. intravenous, intramuscular, intradermal, intraocular,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. Alternatively, thecarrier may be suitable for non-parenteral administration, such as atopical, epidermal or mucosal route of administration. The carrier maybe suitable for oral administration. Depending on the route ofadministration, the modulator may be coated in a material to protect thecompound from the action of acids and other natural conditions that mayinactivate the compound.

The pharmaceutical compositions of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects.Examples of such salts include acid addition salts and base additionsalts.

Pharmaceutically acceptable carriers comprise aqueous carriers ordiluents. Examples of suitable aqueous carriers that may be employed inthe pharmaceutical compositions of the invention include water, bufferedwater and saline. Examples of other carriers include ethanol, polyols(such as glycerol, propylene glycol, polyethylene glycol, and the like),and suitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. In many cases, it willbe desirable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration.

Pharmaceutical compositions of the invention may comprise additionalactive ingredients.

Also within the scope of the present invention are kits comprisingantibodies, compounds and/or complexes of the invention and instructionsfor use. The kit may further contain one or more additional reagents,such as an additional therapeutic or prophylactic agent as discussedabove.

The compounds identified by the methods and/or antibodies of theinvention and the antibodies of the present invention or formulations orcompositions thereof may be administered for prophylactic and/ortherapeutic treatments.

In therapeutic applications compounds are administered to a subjectalready suffering from a disorder or condition as described above, in anamount sufficient to cure, alleviate or partially arrest the conditionor one or more of its symptoms. Such therapeutic treatment may result ina decrease in severity of disease symptoms, or an increase in frequencyor duration of symptom-free periods. An amount adequate to accomplishthis is defined as a “therapeutically effective amount”.

In prophylactic applications, formulations are administered to a subjectat risk of a disorder or condition as described above, in an amountsufficient to prevent or reduce the subsequent effects of the conditionor one or more of its symptoms. An amount adequate to accomplish this isdefined as a “prophylactically effective amount”. Effective amounts foreach purpose will depend on the severity of the disease or injury aswell as the weight and general state of the subject.

A subject for administration may be a human or non-human animal. Theterm “non-human animal” includes all vertebrates, e.g., mammals andnon-mammals, such as non-human primates, sheep, dogs, cats, horses,cows, chickens, amphibians, reptiles, etc. An embodiment of theinvention is administration to humans.

A compound or pharmaceutical composition of the invention may beadministered via one or more routes of administration using one or moreof a variety of methods known in the art. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. Examples of routes of administrationfor compounds or pharmaceutical compositions of the invention includeintravenous, intramuscular, intradermal, intraocular, intraperitoneal,subcutaneous, spinal or other parenteral routes of administration, forexample by injection or infusion. The phrase “parenteral administration”as used herein means modes of administration other than enteral andtopical administration, usually by injection. Alternatively, compound orpharmaceutical composition of the invention can be administered via anon-parenteral route, such as a topical, epidermal or mucosal route ofadministration. The compound or pharmaceutical composition of theinvention may be for oral administration.

A suitable dosage of a compound or pharmaceutical composition of theinvention may be determined by a skilled medical practitioner. Actualdosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A suitable dose may be, for example, in the range of from about 0.01μg/kg to about 1000 mg/kg body weight, or from about 0.1 μg/kg to about100 mg/kg body weight, of the patient to be treated. As a non-limitingexample, a suitable dosage may be from about 1 μg/kg to about 10 mg/kgbody weight per day or from about 10 μg/kg to about 5 mg/kg body weightper day.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single dose may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Administration may be in single or multiple doses. Multiple doses may beadministered via the same or different routes and to the same ordifferent locations. Alternatively, doses can be via a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency may vary depending on the half-life of theantagonist in the patient and the duration of treatment desired.

As mentioned above, compounds or pharmaceutical composition of theinvention may be co-administered with one or other more othertherapeutic agents. For example, the other agent may be an analgesic,anesthetic, immunosuppressant or anti-inflammatory agent.

Combined administration of two or more agents may be achieved in anumber of different ways. Both may be administered together in a singlecomposition, or they may be administered in separate compositions aspart of a combined therapy. For example, the one may be administeredbefore, after or concurrently with the other.

The following examples are presented below so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the methods and compositions of the invention. Theexamples are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1—Synthesis of Compounds

Synthesis of compound (1) is disclosed in WO 2013/186229 (Example 44).

Synthesis of compound (2) is disclosed in WO 2013/186229 (Example 89).

Synthesis of compound (3) is disclosed in WO 2014/009295 (Example 129).

Synthesis of compound (4) is disclosed in WO 2014/009295 (Example 173).

Synthesis of compound (5) is disclosed in WO 2014/009295 (Example 319).

Synthesis of compound (6) is disclosed in WO 2013/186229 (Example 490).

Synthesis of compound (7) is disclosed in WO 2013/186229 (Example 156).

Example 2—Antibody Derivation

Following the immunisation of 5 Sprague Dawley rats with human TNFα incomplex with the benzimidazole compound (1), immune B cells werecultured in 96-well plates to induce clonal expansion and antibodysecretion (Tickle, S. et al., High throughput screening for highaffinity antibodies Journal of Laboratory Automation 2009 14: 303-307).Culture supernatants were screened for IgG antibodies preferentiallybinding to human TNFα in complex with compound (1) (at a 50 fold molarexcess), compared to apo human TNFα, in a homogeneous bead-based FMATassay. Human TNFα (+/− compound (1)) was presented on bead surfaces(superavidin-coated Bangs Beads, catalogue number CP01N) by a capturesystem using a human TNF-Receptor I-Fc fusion protein (R&D Systemscatalogue number 372-R1-050), bound with biotinylated anti-human Fc(Jackson catalogue number 109-066-098).

Antibodies which demonstrated preferential binding to the TNFα-compound(1) complex were termed ‘conformation-selective’ and were taken forwardfor cloning. The Fluorescent Foci method (U.S. Pat. No. 7,993,864/EuropeEP1570267B1) was used to identify and isolate antigen-specific B cellsfrom positive wells, and specific antibody variable region genes wererecovered from single cells by reverse transcription (RT)-PCR.

The amino acid sequences of two representative antibodies, CA185_01974and CA185_01979, which demonstrated conformation-selective binding toboth human and mouse TNFα+compound are shown below:

CA185_01974.0 (VR0001837) Light chain variable region (LCVR) (CDRsunderlined) SEQ ID NO: 7DIQMTQSPASLPASPEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSASRSGTQYSLKISRLQVEDFGIFYCLQGQSTPYTFGAGT KLELKHeavy chain variable region (HCVR) (CDRs underlined) SEQ ID NO: 8DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEAYG YNSNWFGYWGQGTLVTVSSCA185_01979.0 (VR0001842) Light chain variable region (LCVR) (CDRsunderlined) SEQ ID NO: 22DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPHLLIYGTTSLADGVPSRFSGSRSGTQYSLKISGLQVADIGIYVCLQAYSTPFTFGSGT KLEIKHeavy chain variable region (HCVR) (CDRs underlined) SEQ ID NO: 23EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGY NAYHFDYWGRGVMVTVSS

Example 3—Potential Epitope of the Derived Antibodies

Given the ability of the rat-derived antibodies to bind to both humanand mouse TNFα in the presence of compounds, detailed analysis of rat,mouse and human amino acid sequences, together with X-ray crystalstructures of TNFα, was undertaken to see if a likely epitope could bedetermined.

RAt UniProt P16599 (SEQ ID NO: 32)         10         20         30         40         50         60MSTESMIRDV ELAEEALPKK MGGLQNSRRC LCLSLFSFLL VAGATTLFCL LNFGVIGPNK         70         80         90        100        110        120EEKFPNGLPL ISSMAQTLTL RSSSQNSSDK PVAHVVANHQ AEEQLEWLSQ RANALLANGM       130        140        150        160        170        180DLKDNQLVVP ADGLYLIYSQ VLFKGQGCPD YVLLTHTVSR FAISYQEKVS LLSAIKSPCP       190        200        210        220        230KDTPEGAELK PWYEPMYLGG VFQLEKGDLL SAEVNLPKYL DITESGQVYF GVIALMouse UniProt P06804 (SEQ ID NO: 33)        10         20         30         40         50         60MSTESMIRDV ELAEEALPQK MGGFQNSRRC LCLSLFSFLL VAGATTLFCL LNFGVIGPQR        70         80         90        100        110        120DEKFPNGLPL ISSMAQTLTL RSSSQNSSDK PVAHVVANHQ VEEQLEWLSQ RANALLANGM       130        140        150        160        170        180DLKDNQLVVP ADGLYLVYSQ VLFKGQGCPD YVLLTHTVSR FAISYQEKVN LLSAVKSPCP       190        200        210        220        230KDTPEGAELK PWYEPIYLGG VFQLEKGDQL SAEVNLPKYL DFAESGQVYF GVIALHuman UniProt P01375 (SEQ ID NO: 34)        10         20         30         40         50         60MSTESMIRDV ELAEEALPKK TGGPQGSRRC LFLSLFSFLI VAGATTLFCL LHFGVIGPQR        70         80         90        100        110        120EEFPRDLSLI SPLAQAVRSS SRTPSDKPVA HVVANPQAEG QLQWLNRRAN ALLANGVELR       130        140        150        160        170        180DNQLVVPSEG LYLIYSQVLF KGQGCPSTHV LLTHTISRIA VSYQTKVNLL SAIKSPCQRE       190        200        210        220        230TPEGAEAKPW YEPIYLGGVF QLEKGDRLSA EINRPDYLDF AESGQVYFGI IAL

From alignments and comparison of the rat, mouse and human TNFα UniProtsequences, examples of where the rat amino acid sequence differs fromthe human, and where the human and mouse sequences are identical in themature, cleaved product include N168, I194, F220 and A221 (residues andnumbering from the human sequence).

These residues are highlighted on the crystal structure of human TNFα(1TNF) (FIG. 1). It is possible that any of these amino acids areincluded in the epitope targeted by the antibodies CA185_01974 andCA185_01979.

Following cloning of the antibody variable regions into mouse IgG andmouse Fab (no-hinge) vectors, the conformation-selective nature of thebinding of antibodies CA185_01974 and CA185_01979 was confirmed, using avariety of test compounds bound to TNFα, in HPLC, BIAcore, ELISA andcell-based assays.

A more comprehensive analysis of the epitope for CA185_01979 ispresented in Examples 9 and 10 below.

Example 4—High Performance Liquid Chromatography (HPLC) to DetermineAntibody Characteristics

Specific binding of mouse Fab fragments was demonstrated by complexformation between CA185_01974 and human TNFα complexed with compound (1)using size exclusion chromatography. Results are shown in FIG. 2. Asshown in this Figure, with a 0.5× molar excess of Fab the predominantpeak corresponds to bound Fab and trimer-compound complex (althoughthere is a small peak showing the presence of some trimer-compoundcomplex not bound to Fab). At a 1.0× molar excess of Fab there is singlehigher molecular weight peak corresponding to Fab bound totrimer-compound complex. At 1.5× and 2× molar excesses of Fab, there isa growing lower molecular peak corresponding to unbound Fab.

The stoichiometry was therefore determined to be 1 Fab: 1 TNFα trimer,with excess Fab appearing at 1.5× and 2× molar excess.

Binding of CA185_01979 to human TNFα complexed with compound (1) wasalso investigated using size exclusion chromatography. Results are shownin FIG. 3. As for CA185_01974, the stoichiometry was determined to be 1Fab: 1 TNFα trimer, with excess Fab appearing at 1.5× and 2× molarexcess.

Example 5—BIAcore Assays to Determine Antibody Characteristics

Surface plasmon resonance was performed at 25° C. using a BIAcore T200(GE Healthcare). Anti-Mouse Fc (Jackson 115-006-071) was immobilised ona CMS Sensor Chip (GE Healthcare) via amine coupling chemistry to acapture level of ˜6000 response units. HBS-EP buffer (10 mM HEPES pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.05% (v/v) surfactant P20-GEHealthcare)+1% DMSO was used as the running buffer. A 10 μl injection ofeach IgG at 1 μg/ml was used for capture by the immobilised anti-mouseFc to create the TNFα-binding surface. Human or mouse TNFα (in-house) at50 nM was pre-incubated with 2 μM compound in HBS-EP+ (1% DMSO) for 5hours.

A 3 minute injection of human or mouse TNFα+/−test compound was passedover each captured IgG at a flow rate of 30 μl/min. The surface wasregenerated at a flow-rate of 10 μl/min by a 60 s injection of 40 mMHCl×2 and a 30 s 5 mM NaOH. Double referenced background subtractedbinding curves were analysed using the T200 Evaluation software (version1.0) following standard procedures. Kinetic parameters were determinedfrom the fitting algorithm.

The kinetic binding data for human and mouse TNFα in the presence andabsence of test compounds from two chemical series are shown in Tables 1and 2 below.

TABLE 1 BIAcore data with human TNFα Antibody Human TNFα ka (M⁻¹s⁻¹) kd(s⁻¹) KD (M) CA185_01974 +compound (2) 4.2 × 10⁵ 3.9 × 10⁻⁵ 9.4 × 10⁻¹¹CA185_01974 +compound 1 3.2 × 10⁵ 3.8 × 10⁻⁵ 1.2 × 10⁻¹⁰ CA185_01974 apo6.6 × 10⁴ 1.3 × 10⁻³ 1.9 × 10⁻⁸  CA185_01979 +compound (2) 5.7 × 10⁵ 3.3× 10⁻⁵ 5.8 × 10⁻¹¹ CA185_01979 +compound (1) 4.7 × 10⁵ 1.6 × 10⁻⁵ 3.4 ×10⁻¹¹ CA185_01979 apo 1.1 × 10⁵ 7.1 × 10⁻⁴ 6.7 × 10⁻⁹ 

Both CA185_01974 and CA185_01979 demonstrated >2 log selective bindingfor compound-distorted human TNFα, with representative test compoundsfrom two chemical series.

TABLE 2 BIAcore data with mouse TNFα Antibody Mouse TNFα ka (M⁻¹s⁻¹) kd(s⁻¹) KD (M) CA185_01974 +compound (2) 6.7 × 10⁴ 4.8 × 10⁻⁵ 7.1 × 10⁻¹⁰CA185_01974 +compound (1) 5.8 × 10⁴ 8.8 × 10⁻⁵ 1.5 × 10⁻⁹  CA185_01974apo 4.2 × 10⁴ 4.9 × 10⁻³ 1.2 × 10⁻⁷  CA185_01979 +compound (2) 1.9 × 10⁵3.5 × 10⁻⁵ 1.9 × 10⁻¹⁰ CA185_01979 +compound (1) 1.6 × 10⁵ 6.3 × 10⁻⁵3.8 × 10⁻¹⁰ CA185_01979 apo 7.2 × 10⁴ 2.0 × 10⁻³ 2.7 × 10⁻⁸ 

Both CA185_01974 and CA185_01979 demonstrated >1.5 and >2 log selectivebinding for compound-distorted mouse TNFα, with representative testcompounds from two chemical series.

Example 6—ELISAs to Determine Antibody Characteristics

A sandwich ELISA was developed to measure the concentration of TNFαbound to compounds of the invention, using antibody CA185_01974.0 thatspecifically detects the conformation of TNFα when in complex with thesecompounds. Briefly, a microtitre plate was coated with CA185_01974.0 toimmobilise TNFα in complex with a test compound. TNFα was incubatedovernight at 2-8° C. with a 50× molar excess of the test compound.Following this overnight incubation, TNFα was serially diluted in neathuman plasma depleted of endogenous TNFα, in the presence ofheterophilic antibody blockers, and added to the coated plate. Curveswere generated with a concentration range of 0.78 pg/ml-50 pg/ml TNFα. Abiotinylated polyclonal anti TNFα antibody was used to detect boundTNFα, with streptavidin-peroxidase and TMB substrate to give acolorimetric signal. Sensitivity of the assay was increased with the useof tyramide signal amplification, using the ELAST kit from Perkin Elmer,as an additional step between streptavidin-peroxidase and the substrate.

An ELISA was also developed to measure total TNFα (free TNFα+TNFα incomplex with a test compound) in parallel. For this assay the coatingantibody was replaced with a commercial anti-TNFα polyclonal antibody(Invitrogen AHC3812). The sample incubation time was also increased to 3hours. All other steps were identical to the conformation-specificassay. This enables the amount of TNFα in complex with a test compoundto be calculated as a proportion of total TNFα.

Results for the total TNFα ELISA with compounds (3), (4) and (5) areshown in FIG. 4.

Results of the conformation specific TNFα ELISA with CA185_01974.0 andcompounds (3), (4) and (5) are shown in FIG. 5. Apo TNFα gave no signalin this assay, demonstrating the specific nature of the binding ofantibody CA185_01974 to compound-bound TNFα. The antibody was able torecognise TNFα bound by a variety of test compounds from differentchemical series.

Example 7—Cell-Based Assays to Determine Antibody Characteristics

Recombinant antibodies were also tested for binding tocompound-distorted TNFα in a FACS assay using human embryonic kidney(HEK) JumpIn cells, which overexpress TNF-RI after induction withdoxycycline at 1 μg/ml for 2.5 hours. HEK cells were trypsinised andincubated for 2 h in medium to allow recovery of digested TNFRI levels.Human TNFα at 2 μg/mL was pre-incubated with 40 μM compound (1) or 0.4%DMSO for 1 h at 37° C. The pre-incubation mix was added to the cells for1 h on ice (dilution 1:4, final concentrations: 0.5 μg/mLhuman-TNFα+/−10 compound (1) or 0.1% DMSO). Cells were washed, fixed(1.5% PFA) and stained with 1 or 10 μg/mL antibody for 1 h on ice.(Secondary antibody: anti-mouse-Alexa488), before analysis forreceptor-bound TNFα.

As shown in FIG. 6, FACS histogram plots of staining with CA185_01974and CA185_01979 at 1 and 10 μg/ml demonstrate that the antibodies onlyrecognise TNFα which has been pre-incubated with compound (1). There isno staining with the DMSO control.

In addition, specific binding of CA185_01974 and CA185-01979 Fabfragments was demonstrated with compound-distorted membrane-bound TNFα.An engineered NS0 cell line, which overexpresses membrane TNFα, due toknock-out of the TACE cleavage site was incubated with 0.001-10 μMcompound (1) or 0.1% DMSO for 1 h at 37° C. Cells were washed, fixed andstained with antibody Fab fragments at 0.01 or 0.1 μg/ml for 1 hour onice. (Secondary antibody was anti-mouse Fab-DyeLight488 from JacksonImmunoResearch).

FACS histogram plots of staining with CA185_01974 (FIG. 7) andCA185_01979 Fab fragments demonstrate that the antibodies only recogniseTNF which has been pre-incubated with compound (1). There is no stainingwith the DMSO controls.

Example 8—Antibody CA185_01974, Shows a 300-Fold Selectivity for HumanTNF-Compound (4) Complex

Compound (4) was incubated with human and cynomolgus TNF and titratedover mouse full length antibody CA185_01974 to determine an accurateaffinity value. The experiment included the following controls: (i)human or cynomolgus TNF+DMSO over 1974; (ii) human or cynomolgusTNF+DMSO over no antibody; and (iii) human or cynomolgus TNF+compound(4) over no antibody. Each sample and control was carried out induplicate and used four concentrations in each replicate.

As shown in FIGS. 8 and 9, background binding of hTNF+compound (4) andcTNF+compound (4) increased by 5-10 RU over the course of the assay.This is seen in the higher response of h/cTNF+compound (4) binding toCA185_01974 in the second duplicate. Binding of hTNF and cTNF in theabsence of compound (4) was consistently very low.

Kinetics of hTNF+DMSO binding mouse full length IgG CA185_1974_P8 wasvery similar in this assay to previous single concentration analysis.Affinity of cynomolgus TNF for mouse full length IgG CA185_1974_P8 issimilar, however the kinetics differ.

Table 3 gives the kinetics of each analyte binding to CA185_1974_P8.Table 4 gives the average values and the fold difference +/−compound (4)of TNF kinetics for CA185_1974_p8. FIG. 8 gives the sensograms of bothduplicates of cTNF+/−compound (4). FIG. 9 gives the sensograms of bothduplicates of hTNF+/−compound (4).

TABLE 3 Binding kinetics of hTNF and cTNF +/− compound (4) to theCA185_1974_P8 antibody Duplicate Antibody Analyte ka (1/Ms) kd (1/s) KD(M) KD (pM) 1 CA185_1974_P8 cyno TNF 1.03E+05 1.87E−03 1.83E−08 18270 2CA185_1974_P8 cyno TNF 1.25E+05 1.92E−03 1.54E−08 15350 1 CA185_1974_P8cyno TNF + 1.84E+05 1.46E−05 7.91E−11 79.1 compound (4) 2 CA185_1974_P8cyno TNF + 2.01E+05 2.06E−05 1.03E−10 103 compound (4) 1 CA185_1974_P8human TNF 8.02E+04 1.77E−03 2.21E−08 22100 2 CA185_1974_P8 human TNF1.05E+05 1.67E−03 1.59E−08 15900 1 CA185_1974_P8 human TNF + 3.06E+051.00E−05 3.27E−11 32.7 compound (4) 2 CA185_1974_P8 human TNF + 3.07E+052.73E−05 8.88E−11 88.8 compound (4)

TABLE 4 Average values and fold differences +/− compound (4) of hTNF andcTNF kinetics for CA185_1974_p8. Average of duplicates ka (1/Ms) kd(1/s) KD (M) KD (pM) cyno TNF 1.14E+05 1.90E−03 1.68E−08 16810 cynoTNF + compound (4) 1.93E+05 1.76E−05 9.09E−11 90.92 Fold difference 1.69107.81 184.89 184.89 human TNF 9.27E+04 1.72E−03 1.90E−08 19000 humanTNF + compound 3.06E+05 1.86E−05 6.08E−11 60.76 (4) Fold difference 3.3192.43 312.70 312.70

Example 9—Compounds and Complexes of Ma et al (2014) and Silvian et al(2011) have Different Characteristics to Those of the Present Invention

As described on page 12458 of Ma et al. (2014) JBC 289:12457-12466, C87was discovered through virtual screening by attempting to find moleculeswhich fit the space occupied by a 7 amino-acid peptide fromloop2/domain2 of TNFR1 in its interaction with the external surface ofTNFβ. The C87 compound from Ma et al. and the BIO8898 compound fromSilvian et al. (2011) ACS Chemical Biology 6:636-647 were tested by thepresent inventors.

Summary of Findings

The Biacore observations described in Ma et al. for C87 could not berepeated.

No evidence of TNF specific inhibition in cells was observed.

Additionally C87 was not observed to bind by mass spectrometry, which issensitive to millimolar affinities.

Extensive crystallography trials only produced apo-TNF (TNF withoutcompound).

In the fluorescence polarisation (FP) assay, C87 showed no significantinhibition above the interference level of the compound with thefluorescent read-out.

Thermofluor, which measures stabilisation of the thermal meltingtemperature of TNFα, did show a small stabilisation for C87.

In summary, no evidence was found that C87 binds in the centre of thetrimer. The overwhelming majority of the data suggested no directinteraction with TNFα. BIO8898 was also found not to bind to TNFα.

Cells—TNF Induced HEK NFKB Reporter Gene Assay

C87 was pre-incubated with TNFα for 1 hour prior to the addition toHEK-293 cells stably transfected with SEAP under the control of NFκB. Anappropriate counter-screen was also tested in order to detect non-TNFrelated (off target) activity. The assay was incubated overnight beforeinhibition was measured compared to 100% blocking by a control compound.The maximum C87 concentration was 10,000 nM, with a 3-fold serialdilution.

No inhibitory effect could be detected that could not be attributed tooff-target activity.

Biacore

TNF was immobilised using an avi-tag linker and C87 was passed over thechip. In one experiment, a dose response of C87 from a highestconcentration of 10 μM was performed. No binding was observed.

In a second experiment, the flow rate of C87 passing over the chip wasreduced. A small shift was observed but overall binding was negligible.

The binding of C87 to TNF described in Ma et al was likely to besuper-stoichiometric based on the RU value on the Y-axis. At standardTNF density on the chip this value was in the region of thirty timeshigher than expected for simple 1:1 binding.

In another experiment, BIO8898 was tested against the immobilisedsoluble form of CD40L and the soluble form of TNFα by SPR on a Biacore4000 machine. A geomean IC50 of 17 μM was determined for binding againstCD40L whereas no binding was detected at a concentration of up to 100 μMfor TNFα in this assay.

Mass Spectrometry

There was no evidence of C87 binding to human TNFα (20 μM) at aconcentration of 400 μM. A species of lower molecular weight (˜473 Daappears to bind at less than 5% occupancy). C87 has a molecular weightof 503 Da. Based on the occupancy at a concentration of 400 μM, anaffinity of the low molecular weight species in excess of 1 mM ispredicted.

Crystallography

Overall a large effort was put into crystallising C87 with TNFα,including testing conditions that routinely work with compoundsdescribed in the present application. This comprised setting up a largenumber of crystallization trials at different ligand concentrations,different protein concentrations, and different soaking times. A fewcrystals were observed that, on analysis, proved to be salt or TNF withno compound.

Fluorescent Polarization (FP)

C87 was preincubated with TNFα for 1 hour prior to assay against thefluorescent compound (probe). Competition with the fluorescent compoundeither directly (binding at the same site) or indirectly (disruptingTNF) is detected by a reduction in FP.

Extrapolation of the inhibition curve produced an IC50 of about 100 μM.Fluorescence quenching was, however, observed at the highestconcentrations of inhibitor which, when subtracted, resulted innegligible inhibition of C87 in this assay.

Thermofluor

Thermofluor measures the change of melting temperature (Tm) of TNFα dueto compound either stabilising or disrupting the protein. Astabilization effect of 3.8° C. was observed at a concentration of 500μM C87, suggesting the possibility of weak binding, which may not bespecific.

Example 10—hTNFα and Fab1974 Complex Structure 1) Protein Expression andPurification

The soluble form of human TNFα (CID2043, UniProt P01375, residues77-233) was expressed as a fusion protein in E. coli and had the finalsequence:

(SEQ ID NO: 35) SVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYF GIIAL

The initial “S” of SEQ ID NO: 35 is a cloning artefact and not part ofthe native sequence of the TNF. The residue numbering of SEQ ID NO: 35therefore starts from V i.e. V1, R2, S3 etc. SEQ ID NO: 36 representsSEQ ID NO: 35, but without this initial “S” residue i.e.

(SEQ ID NO: 36) VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFG IIAL

Cells were pre-cultured at 37° C. in rich media, induced with theaddition of 0.1% arabinose and allowed to express overnight at 25° C. invector pEMB54. This vector introduces a cleavable N-terminalHis6Smt-tag. The cells were lysed and purified by Ni-NTA chelatechromatography. The fusion protein was eluted with buffer containingimidazole and cleaved by the addition of protease. The final cleavedTNFα protein was purified by a subtractive Ni chelate chromatographystep to remove the fusion tag and further purified by size exclusionchromatography to remove the remaining impurities. The final TNFαproduct was typically concentrated to 20.0 mg/ml and flash frozen inliquid nitrogen.

The extracellular domain of human TNFR1 (CID5602, UniProt P19438,residues 42-184) with N54D and C182S mutations was expressed as asecreted protein in baculovirus infected insect cells and has the finalsequence:

(SEQ ID NO: 37) GSVCPQGKYIHPQDNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSSSN

The initial “G” of SEQ ID NO: 37 is a cloning artefact and not part ofthe native sequence of the TNFR1. The residue numbering of SEQ ID NO: 37therefore starts from S i.e. 51, V2, C3 etc. SEQ ID NO: 38 representsSEQ ID NO: 37, but without this initial “G” residue i.e.

(SEQ ID NO: 38) SVCPQGKYIHPQDNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSSSN

The fusion protein plasmid was cloned into the pEMB50 expression vector,which encodes a cleavable N-terminal secretion signal and His-taggedfusion protein. Virus was generated using the baculovirus expressionsystem. Infected insect cells secreted the fusion protein into themedia. The fusion protein was purified by Ni-NTA chelate chromatographyand eluted from the Ni column using an imidazole gradient. The elutedprotein was cleaved with protease to release the N-terminal His-fusiontag. The cleaved TNFR1 was subsequently purified by a subtractive Nichelate chromatography step and further purified by size exclusionchromatography. The final TNFR1 product was typically concentrated to10.0 mg/ml and flash frozen in liquid nitrogen.

To form the complex, 333 μl of purified human TNFα at 300 mM was mixedwith 4,567 μl of SEC buffer (10 mM HEPES, pH 7.5, 150 mM NaCl) and 100μL of compound (2) (10 mM in DMSO, approximately 10 molar excess) andincubated at 4° C. overnight. The following day, the complex was formedby adding 4080 μl of SEC buffer to 700 μl TNFR1, 5,000 μl ofTNFα/UCB1478733 mix, and 220 μl Fab1974 at 500 mM; Total volume of thereaction was 10 ml with a final molar ratio of 3 TNFα monomers(equivalent to 1 trimer): 2.5 TNFR1 receptors: 1.2 Fab. The ternarycomplex (cytokine, ligand, receptor, and Fab) was incubated for 1 hourat 4° C. The sample was then concentrated to 1.5 ml and was loaded in asingle injection on Superdex 200 16/600 size exclusion column (120 ml)that was pre-equilibrated with 10 mM HEPES pH 7.5, 150 mM NaCl. Peakfractions of the ternary complex were selected and concentrated to 13.7mg/ml and immediately used in crystallization trials.

2) Crystallization

The ternary complex was crystallized by sitting drop vapor diffusion bymixing 0.5 μl of complex with 0.5 μl of Wizard III/IV: 0.1M HEPES,pH7.0, 10% PEG6,000 (condition B8) over 80 μl of the samecrystallization solution. Crystals were harvested for data collectionapproximately 2 months after initial set up. They were cryo-protected inglycerol performed in 5-10-15% steps, then frozen directly in liquidnitrogen for data collection on Jun. 8, 2016 at Advanced Photon Sourceat Argonne National Laboratory, Life Sciences Collaborative Access Team(LS-CAT), beamline 21-ID-F.

3) Structure Determination

The structure of the human TNFα (CID2043), human TNFR1 (CID5602), andFab1974 complex with ligand compound (2) was solved by molecularreplacement using Phenix.Phaser with input models based upon previouslydetermined structure of human TNFα, human TNFR1, Fab1979 bound withUCB1478733 and a separately determined structure of the isolatedFab1974. Data were integrated in XDS and scaled using XSCALE 1. Initialstructure determination and refinement used data to 3.00 Å resolutionfrom a single crystal. Iterative manual model building using Coot 3 andin Phenix.Refine 2 continued until R and Rfree reached R=0.201,Rfree=0.259. Model quality was validated using Coot and MolProbity 4.Final data processing and refinement statistics are listed in Table 1.

4) References

-   1. Kabsch, W., 2010. Xds. Acta Crystallographica Section D:    Biological Crystallography, 66(2), pp. 125-132.-   2. Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B.,    Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J.,    Grosse-Kunstleve, R. W. and McCoy, A. J., 2010. PHENIX: a    comprehensive Python-based system for macromolecular structure    solution. Acta Crystallographica Section D: Biological    Crystallography, 66(2), pp. 213-221.-   3. Emsley, P., Lohkamp, B., Scott, W. G. and Cowtan, K., 2010.    Features and development of Coot. Acta Crystallographica Section D:    Biological Crystallography, 66(4), pp. 486-501.-   4. Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A.,    Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S.    and Richardson, D. C., 2010. MolProbity: all-atom structure    validation for macromolecular crystallography. Acta    Crystallographica Section D: Biological Crystallography, 66(1), pp.    12-21.    Data Collection and Refinement Statistics.

Parameter Overall (Highest Shell) Data collection Dataset 1 Crystal ID272787b8 Beamline APS 21-ID-F Protein Human TNFα, Human TNFR1, Fab1974Ligand Compound (2) Collection date Jun. 8^(th), 2016 Oscillation width(°) 1.0 Frames 150 Exposure (sec) 1.5 Distance (mm) 410 Wavelength (Å)0.97872 Data processing Overall (Highest shell) Space Group P 4₁ 2₁ 2Unit cell (Å, °) a = b = 99.51, c = 311.35; α = β = γ = 90 Resolution(Å) 50.00-3.00 (3.08-3.00) I/σ 14.61 (2.27) Completeness (%) 99.90(99.97) Rpim 0.049 (0.38) R_(merge) (%) 16.4 (127.9) Reflections(unique) 32,358 (3,171) Multiplicity 12.1 (12.4) Refinement statisticsResolution (Å) 47.39-3.00 (3.11-3.00) R_(work)/R_(free) overall22.29/25.56 (33.43/37.58) No. atoms Protein 8396 Ligand 109 Water 20 ADP(Å²) Protein 69.69 Ligand 64.03 Water 47.65 r.m.s. deviations Bondlengths (Å) 0.04 Bond angles (°) 0.96 Ramachandran outliers (%) 0.09Ramachandran favored (%) 97.24 Molprobity score 1.30; 100^(th)percentile* (N = 3130, 3.00 Å ± 0.25 Å) Refined by: David Fox III PeerReviewed by: Deborah G. Conrady *100th percentile is the best amongstructures of comparable resolution; 0th percentile is the worst.

6) Structure Discussion and Figures

FIG. 11 shows the structure of the Fab1974 bound to a complex of humanTNFα asymmetric trimer, compound (2), and 2 human TNFR1 receptors.

The main contact between TNFα and the Fab is via the heavy chain and thedisplaced TNFα monomer A. Heavy chain CDRs 2 and 3 form a contact withthe outer face of monomer A and CDR3 protrudes deeply into the opencleft of the distorted A/C receptor binding site. The positioning ofCDR3 in this cleft results in the only contact between the CA1974 heavychain and TNFα monomer C where pi stacking occurs between Tyr103 of CDR3and Tyr115 of monomer C.

The light chain of CA1974 also forms a substantial contact with TNFα,bridging the distorted A/C receptor binding cleft. The light chain CDR2contacts with monomer C exclusively, towards the bottom of the cleft.Only two residues of CDR1 contact TNFα, one binding monomer C and theother monomer A, on the inner faces of the cleft. Light chain CDR3associates solely with monomer A, interacting with residues towards thetop of the cleft.

FIG. 12 shows the same structure as FIG. 11, but with a symmetric trimer(i.e. an undistorted trimer in the absence of ligand) modelled incomputationally. With this symmetric trimer, the A chain of the trimerretains the interaction with the Fab fragment. However, there is asteric clash between the Fab fragment and the C chain of the trimer.

Example 11—Identification of Epitope Residues

The program NCONT (part of the CCP4 crystallography suite) was used toidentify residues between the Fab1974 and the TNF asymmetric trimer,within a distance of 4.0 Å. Any atom in TNFα from a residue within thatdistance was identified as an epitope residue. Defining an epitope asatoms of the antigen which are within 4.0 Å of antibody atoms is routinein the art (see for example Andersen et al (2006), Protein Science, 15,2258-2567).

Results of this experimentally determined epitope are presented in FIG.13. Residues on the A chain of TNFα identified in this way were T77,T79, I83, T89, K90, V91, N92, L93, L94, S95, A96, I97, K98, E135, I136,N137 and R138. Residues on the C chain of TNFα identified in this waywere L63, K65, Q67, P113, Y115, D143, A145, E146 and Q149. Residuenumbering is based on SEQ ID NO: 36 (the soluble human TNFα sequencelacking the “S” cloning artefact).

The following additional A chain residues (also potentially forming partof a wider epitope) within a 5.0 Å proximity were identified: Q27, Q47,S81, Q88, Y119, R131 and S133. The following additional C chain residue(also potentially forming part of a wider epitope) within a 5.0 Åproximity was identified: P117. Again, residue numbering is based on SEQID NO: 36. These 4.0 and 5.0 Å residues are summarised in the tablebelow, together with the residues on Fab 1974:

TNF-A TNF-C Fab1974-L Fab1974-H TNF-A TNF-C Fab1974-L Fab1974-H  (4 Å)(4 Å) (4 Å) (4 Å) (5 Å) (5 Å) (5 Å) (5 Å) T77 L63 N31 T28 Q27 L63 G30T28 T79 K65 W32 S30 Q47 K65 N31 S30 I83 Q67 Y49 A31 T77 Q67 W32 A31 T89P113 T52 Y33 T79 P113 Y49 Y33 K90 Y115 S53 N52 S81 Y115 T52 N52 V91 D143R66 Y53 I83 P117 S53 Y53 N92 A145 G91 F59 Q88 D143 L54 D54 L93 E146 Q92Y101 T89 A145 R66 N57 L94 Q149 S93 G102 K90 E146 G91 F59 S95 T94 Y103V91 Q149 Q92 E99 A96 Y96 N104 N92 S93 Y101 I97 N106 L93 T94 G102 K98 L94Y96 Y103 E135 S95 N104 I136 A96 S105 N137 I97 N106 R138 K98 Y119 R131S133 E135 I136 N137 R138

Example 12—Assessment of 1974 as a Target Occupancy Reagent

The ability of CA1974 to detect a range of different recombinant humanTNFα concentrations pre-incubated with saturating levels of smallmolecule was tested. Pre-incubation of TNFα with excess compound wasachieved by mixing 10 μs/ml TNFα with either 1% (v/v) DMSO or 10 μMcompound (2) in 0.1% (w/v) BSA/PBS, resulting in an approximately50-fold molar excess of small molecule inhibitor over TNFα.

Following overnight incubation the complex was diluted to a range ofconcentrations in human plasma that had been depleted of endogenous TNFαby immunoprecipitation using an in-house anti-TNFα monoclonal antibody.Small molecule-bound TNFα was measured using a sandwich ELISA. A 96 wellELISA plate (Nunc) was coated with 0.1 μg/ml CA1974 in PBS overnight at4° C. After washing three times in wash buffer (0.05% (v/v)Tween-20/PBS) the plate was blocked in 2% (w/v) BSA/PBS for at least 1 hat RT. 100 μl of each sample was loaded to the blocked ELISA plate andincubated for 30 mins at RT with shaking at 200 rpm. The plate waswashed 3 times in wash buffer before adding 100 μl per well ofbiotinylated anti-TNFα detection antibody (BAF210, R&D Systems), at aconcentration of 0.1 μg/ml in 1% (w/v) BSA/wash buffer, for 1 h at RTwith shaking at 200 rpm. Following 3 washes, 100 μl per well of 100ng/ml Streptavidin-horse radish peroxidase (SA-HRP, Sigma) in 1%BSA/wash buffer was added and incubated for 45 mins at RT with shakingat 200 rpm. The plate was washed 3 times in wash buffer before includinga biotin-tyramide (B-T) amplification step to provide additionalamplification, using the ELAST system from Perkin Elmer. B-T solutionwas diluted 1 in 100 in ELAST buffer, 100 μl added per well and theplate incubated for exactly 15 mins at RT with shaking at 200 rpm beforewashing 4 times in wash buffer. An incubation with ELAST SA-HRP wasperformed, followed by 4 washes and the addition of 100 μl TMB (twocomponent, KPL) per well. When appropriate colour change had beenreached, the reaction was stopped by the addition of 100 μl per well of2.5 M sulphuric acid. Absorbance at 450 nm was measured using aMultiskan EX plate reader with Ascent Software version 2.6 (Thermo).

To determine the concentration at which TNFα+excess small moleculeinhibitor could be detected above apo TNFα background, 3 standarddeviations of the TNFα+DMSO OD results was added to the mean of thesevalues. All TNFα+small molecule spike results greater than this valuewere considered to be significantly different to apo TNFα, with aprobability of at least 0.999.

As can be seen FIG. 14, TNFα-small molecule complex could be detected at25 pg/ml and above in neat plasma (with probability of at least 0.999)and there was no background cross-reactivity observed with apo TNFα.This demonstrates that CA1974 may be a useful reagent for themeasurement of target occupancy in complex biological samples.

Example 13—Single Cycle Kinetics of Compound (1) Binding to Human TNFαDirectly Immobilised or Via Capture with CA1974

For single cycle kinetics profiling of compound (1) to either directlyimmobilised TNFα or CA1974-captured TNFα surface plasmon resonance wasperformed at 25° C. using a BIAcore T200 (GE Healthcare). Human TNFα wastethered onto flow-channel (Fc)2 (2000 RU). CA1974 IgG (>10,000 responseunits) was tethered onto Fc3,4 and Fc1 blank immobilisation on a CMSSensor Chip (GE Healthcare) via amine coupling chemistry. The surfacewas allowed to stabilise in HBS-P buffer (10 mM HEPES, pH 7.4, 0.15 MNaCl, 0.0005% (v/v) surfactant P20-GE Healthcare)+5% DMSO was used asthe running buffer. Human TNFα was flowed over Fc4 to a capture level of˜2,000 RU. Compound (1) was flowed over all four flow cells in series(1.875 μM, 3.75 μM, 7.5 μM and 30 μM) at 100 μl/min. Double referencedbackground-subtracted binding curves were produced using the T200Evaluation software (version 1.0) following standard procedures. Resultsare presented in FIG. 15.

As indicated, kinetics change from “slow on-slow off” with directlyimmobilised apo-TNF to “fast on-fast off” when captured via theconformation-selective antibody CA1974. This indicates that the antibodyis able to stabilise an alternative, presumably open conformation whichmight facilitate the identification of new chemical matter which, undernormal circumstances, is entropically disadvantaged.

Interestingly, despite structural models revealing major side chainclashes with the symmetric form of apo TNFα (FIG. 12), both Biacore andSE-HPLC experiments suggested that CA1974 is able to interact with apoTNFα. The affinity of the interaction was typically >100-fold lower thanthat observed with the TNFα-small molecule complex. SE-HPLC suggestedthat CA1974 interacts with a small fraction (21%) of apo TNFα andproduces a complex that has elution characteristics consistent with a1:1 (Fab: apo TNFα trimer) complex. Taken together, these observationssupport the hypothesis that apo TNFα naturally adopts an “open”conformation that allows antibody recognition rather than the antibodybinding to a partial epitope on the symmetric molecule. The lower amountof complex that is present in the SE-HPLC experiment compared to theFab-TNFα-small molecule complex and the lower observed affinity for apoTNFα could imply that this “open” conformation is sampled naturally butexists only transiently and that this form of the apo TNFα is much lessenergetically stable than the small molecule-stabilised form and hencethe antibody interaction is compromised. These findings support theconclusions made in previous molecular dynamic simulation studies andDEER experiments with spin-labelled TNFα which predicts 6% of trimers atany one time adopt a naturally-sampled asymmetric open state. Thepresence of CA1974 in the SEC HPLC experiment likely draws theequilibrium of conformational sampling towards the open state,increasing the percentage of asymmetric trimer.

This highlights how antibodies, which define rarely sampledconformations of target proteins, may facilitate small molecule fragmentscreening by enabling the binding of otherwise entropicallydisadvantaged compounds (FIG. 15) or act as a ‘sensor’ to detect newchemical matter that is able to stabilise predefined inactiveconformations. Conversely, small molecules which stabilise rarelysampled conformations of target proteins, may augment the generation ofantibodies recognising induced neo-epitopes.

Sequence listing (LCDR1 of 1974) SEQ ID NO: 1 QASQDIGN (LCDR2 of 1974)SEQ ID NO: 2 GATSLAD (LCDR3 of 1974) SEQ ID NO: 3 LQGQSTPYT(HCDR1 of 1974) SEQ ID NO: 4 AYYMA (HCDR2 of 1974) SEQ ID NO: 5ASINYDGANTFYRDSVKG (HCDR3 of 1974) SEQ ID NO: 6 EAYGYNSNWFGY(LCVR of 1974) SEQ ID NO: 7DIQMTQSPASLPASPEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSASRSGTQYSLKISRLQVEDFGIFYCLQGQSTPYTFGAGTKLELK (HCVR of 1974) SEQ ID NO: 8DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEAYGYNSNWFGYWGQGTLVTVSS (LCVR DNA of 1974)SEQ ID NO: 9GACATCCAGATGACCCAGTCTCCTGCCTCCCTGCCTGCATCCCCGGAAGAAATTGTCACCATCACATGCCAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGGAGAAACCAGGGAAATCGCCTCAGCTCCTGATCTATGGTGCAACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGCCAGTAGATCTGGCACACAGTACTCTCTTAAGATCAGGAGACTGCAGGTTGAAGATTTTGGAATCTTTTACTGTCTACAGGGTCAAAGTACTCCGTACACGTTTGGAGCTGGGACCAAGCTGGAACTGAAA (HCVR DNA of 1974)SEQ ID NO: 10GACGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGCCTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGTCTGGAGTGGGTCGCATCCATTAATTATGATGGTGCTAACACTTTCTATCGCGACTCCGTGAAGGGCCGATTCACTGTCTCCAGAGATAATGCAAGAAGGAGCCTATACCTACAAATGGACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGTACAACAGAGGCTTACGGATATAACTCAAATTGGTTTGGTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCGAGC (1974 LC kappa full) SEQ ID NO: 11DIQMTQSPASLPASPEEIVTITCQASQDIGNWLSWYQQKPGKSPQLLIYGATSLADGVPSRFSASRSGTQYSLKISRLQVEDFGIFYCLQGQSTPYTFGAGTKLELKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (1974 HC mIgG1 full) SEQ ID NO: 12DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEAYGYNSNWFGYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (1974 HC mFabno hinge full)SEQ ID NO: 13DVQLVESGGGLVQPGRSLKLSCAASGFTFSAYYMAWVRQAPTKGLEWVASINYDGANTFYRDSVKGRFTVSRDNARSSLYLQMDSLRSEDTATYYCTTEAYGYNSNWFGYWGQGTLVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC (1974 LC DNA kappa full) SEQ ID NO: 14GACATCCAGATGACCCAGTCTCCTGCCTCCCTGCCTGCATCCCCGGAAGAAATTGTCACCATCACATGCCAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCGCCTCAGCTCCTGATCTATGGTGCAACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGCCAGTAGATCTGGCACACAGTACTCTCTTAAGATCAGCAGACTGCAGGTTGAAGATTTTGGAATCTTTTACTGTCTACAGGGTCAAAGTACTCCGTACACGTTTGGAGCTGGGACCAAGCTGGAACTGAAACGTACGGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (1974 HC DNA mIgG1 full) SEQ ID NO: 15GACGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGCCTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGTCTGGAGTGGGTCGCATCCATTAATTATGATGGTGCTAACACTTTCTATCGCGACTCCGTGAAGGGCCGATTCACTGTCTCCAGAGATAATGCAAGAAGCAGCCTATACCTACAAATGGACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGTACAACAGAGGCTTACGGATATAACTCAAATTGGTTTGGTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA (1974 HC DNA mFabno hinge full) SEQ ID NO: 16GACGTGCAGCTGGTGGAATCTGGAGGAGGCTTAGTGCAGCCTGGAAGGTCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCACTTTCAGTGCCTATTACATGGCCTGGGTCCGCCAGGCTCCAACGAAGGGTCTGGAGTGGGTCGCATCCATTAATTATGATGGTGCTAACACTTTCTATCGCGACTCCGTGAAGGGCCGATTCACTGTCTCCAGAGATAATGCAAGAAGCAGCCTATACCTACAAATGGACAGTCTGAGGTCTGAGGACACGGCCACTTATTACTGTACAACAGAGGCTTACGGATATAACTCAAATTGGTTTGGTTACTGGGGCCAAGGCACTCTGGTCACTGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCGGCTGTCCTGCAATCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT (LCDR2 of 1979)SEQ ID NO: 17 GTTSLAD (LCDR3 of 1979) SEQ ID NO: 18 LQAYSTPFTF(HCDR1 of 1979) SEQ ID NO: 19 NSYWD (HCDR2 of 1979) SEQ ID NO: 20YINYSGSTGYNPSLKS (HCDR3 of 1979) SEQ ID NO: 21 GTYGYNAYHFDY(LCVR of 1979) SEQ ID NO: 22DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPHLLIYGTTSLADGVPSRFSGSRSGTQYSLKISGLQVADIGIYVCLQAYSTPFTFGSGTKLEIK (HCVR of 1979) SEQ ID NO: 23EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSS (LCVR DNA of 1979)SEQ ID NO: 24GACATCCAAATGACACAGTCTCCTGCCTCCCTGTCTGCATCTCTGGAAGAAATTGTCACCATTACATGCCAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCTCCTCACCTCCTGATCTATGGTACCACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGGCAGTAGATCTGGTACACAGTATTCTCTTAAGATCAGCGGACTACAGGTTGCAGATATTGGAATCTATGTCTGTCTACAGGCTTATAGTACTCCATTCACGTTCGGCTCAGGGACAAAGCTGGAAATAAAA (HCVR DNA of 1979)SEQ ID NO: 25GAGGTGCACCTGGTGGAGTCTGGACCTGGCCTTGTGAAACCCTCACAGTCACTCTCCCTCACCTGTTCTGTCACTGGTTACTCCATCACTAATAGTTACTGGGACTGGATCCGGAAGTTCCCAGGAAATAAAATGGAGTGGATGGGATACATAAACTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCCATTAGTAGAGACACATCGAACAATCAGTTCTTCCTGCAGCTGAACTCTATAACTACTGAGGACACAGCCACATATTACTGTGCACGAGGGACCTATGGGTATAACGCCTACCACTTTGATTACTGGGGCCGAGGAGTCATGGTCACAGTCTCGAGC (1979 LC Kappa full) SEQ ID NO: 26DIQMTQSPASLSASLEEIVTITCQASQDIGNWLSWYQQKPGKSPHLLIYGTTSLADGVPSRFSGSRSGTQYSLKISGLQVADIGIYVCLQAYSTPFTFGSGTKLEIKRTDAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (1979 HC mIgG1 full) SEQ ID NO: 27EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (1979 HC mFabno hinge full) SEQ ID NO: 28EVHLVESGPGLVKPSQSLSLTCSVTGYSITNSYWDWIRKFPGNKMEWMGYINYSGSTGYNPSLKSRISISRDTSNNQFFLQLNSITTEDTATYYCARGTYGYNAYHFDYWGRGVMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC (1979 LC DNA Kappa full) SEQ ID NO: 29GACATCCAAATGACACAGTCTCCTGCCTCCCTGTCTGCATCTCTGGAAGAAATTGTCACCATTACATGCCAGGCAAGCCAGGACATTGGTAATTGGTTATCATGGTATCAGCAGAAACCAGGGAAATCTCCTCACCTCCTGATCTATGGTACCACCAGCTTGGCAGATGGGGTCCCATCAAGGTTCAGCGGCAGTAGATCTGGTACACAGTATTCTCTTAAGATCAGCGGACTACAGGTTGCAGATATTGGAATCTATGTCTGTCTACAGGCTTATAGTACTCCATTCACGTTCGGCTCAGGGACAAAGCTGGAAATAAAACGTACGGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT (1979 HC DNA mIgG1 full) SEQ ID NO: 30GAGGTGCACCTGGTGGAGTCTGGACCTGGCCTTGTGAAACCCTCACAGTCACTCTCCCTCACCTGTTCTGTCACTGGTTACTCCATCACTAATAGTTACTGGGACTGGATCCGGAAGTTCCCAGGAAATAAAATGGAGTGGATGGGATACATAAACTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCCATTAGTAGAGACACATCGAACAATCAGTTCTTCCTGCAGCTGAACTCTATAACTACTGAGGACACAGCCACATATTACTGTGCACGAGGGACCTATGGGTATAACGCCTACCACTTTGATTACTGGGGCCGAGGAGTCATGGTCACAGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA (1979 HC DNA mFabno hinge full) SEQ ID NO: 31GAGGTGCACCTGGTGGAGTCTGGACCTGGCCTTGTGAAACCCTCACAGTCACTCTCCCTCACCTGTTCTGTCACTGGTTACTCCATCACTAATAGTTACTGGGACTGGATCCGGAAGTTCCCAGGAAATAAAATGGAGTGGATGGGATACATAAACTACAGTGGTAGCACTGGCTACAACCCATCTCTCAAAAGTCGAATCTCCATTAGTAGAGACACATCGAACAATCAGTTCTTCCTGCAGCTGAACTCTATAACTACTGAGGACACAGCCACATATTACTGTGCACGAGGGACCTATGGGTATAACGCCTACCACTTTGATTACTGGGGCCGAGGAGTCATGGTCACAGTCTCGAGTGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCGGCTGTCCTGCAATCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGT Rat TNFα SEQ ID NO: 32MSTESMIRDVELAEEALPKKMGGLQNSRRCLCLSLFSFLLVAGATTLFCLLNFGVIGPNKEEKFPNGLPLISSMAQTLTLRSSSQNSSDKPVAHVVANHQAEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLIYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVSLLSAIKSPCPKDTPEGAELKPWYEPMYLGGVFQLEKGDLLSAEVNLPKYLDITESGQVYFGVIAL Mouse TNFα SEQ ID NO: 33MSTESMIRDVELAEEALPQKMGGFQNSRRCLCLSLFSFLLVAGATTLFCLLNFGVIGPQRDEKFPNGLPLISSMAQTLTLRSSSQNSSDKPVAHVVANHQVEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLVYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVNLLSAVKSPCPKDTPEGAELKPWYEPIYLGGVFQLEKGDQLSAEVNLPKYLDFAESGQVYFGVIAL Human TNFα SEQ ID NO: 34MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL Soluble form of human TNFα SEQ ID NO: 35SVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL Soluble form of human TNFαbut excluding the initial “S”of SEQ ID NO: 35, which is a cloning artefact SEQ ID NO: 36VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL Extracellular domain of human TNFR1 with N54D andC182S mutations (including initial “G”, which is a cloning artefact)SEQ ID NO: 37GSVCPQGKYIHPQDNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSSSN Extracellular domain of human TNFR1 with N54D andC182S mutations (excluding initial “G”, which is a cloning artefact)SEQ ID NO: 38SVCPQGKYIHPQDNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSSSN

The invention claimed is:
 1. An antibody comprising: a HCDR1 selectedfrom SEQ ID NOs: 4 and 19; a HCDR2 selected from SEQ ID NOs: 5 and 20;and a HCDR3 selected from SEQ ID NOs: 6 and 21; and a LCDR1 of SEQ IDNO: 1; a LCDR2 selected from SEQ ID NOs: 2 and 17; and a LCDR3 selectedfrom SEQ ID NOs: 3 and
 18. 2. An antibody according to claim 1, which(a) is a humanised antibody; or (b) is a Fab, modified Fab, Fab′,modified Fab′, F(ab′)₂, Fv, single domain antibody or an scFv.
 3. Anantibody according to claim 1, which binds a complex of trimeric TNFαand any one of compounds (1)-(7):

and which optionally has an affinity (K_(D-ab)) of 1 nM or less for thetrimer-compound complex; or an affinity of 200 pM or less.
 4. Theantibody of claim 1, wherein the antibody comprises at least one HCDRselected from SEQ ID NOS:4-6 and at least one LCDR selected from SEQ IDNOS: 1-3.
 5. The antibody of claim 1, wherein the antibody comprises atleast one HCDR selected from SEQ ID NOS:19-21 and at least one LCDRselected from SEQ ID NOS: 1, 17 and
 18. 6. The antibody of claim 1,wherein the antibody comprises at least two HCDRs selected from SEQ IDNOS:4-6 and at least two LCDRs selected from SEQ ID NOS: 1-3.
 7. Theantibody of claim 1, wherein the antibody comprises at least two HCDRsselected from SEQ ID NOS:19-21 and at least two LCDRs selected from SEQID NOS: 1, 17 and
 18. 8. The antibody of claim 1, wherein the antibodycomprises SEQ ID NO:4 for HCDR1, SEQ ID NO:5 for HCDR2 and SEQ ID NO:6for HCDR3 and SEQ ID NO:1 for LCDR1, SEQ ID NO:2 for LCDR2 and SEQ IDNO:3 for LCDR3.
 9. The antibody of claim 1, wherein the antibodycomprises SEQ ID NO:19 for HCDR1, SEQ ID NO:20 for HCDR2 and SEQ IDNO:21 for HCDR3, and SEQ ID NO:1 for LCDR1, SEQ ID NO:17 for LCDR2 andSEQ ID NO:18 for LCDR3.
 10. An antibody comprising (a) a heavy chainvariable region (HCVR) comprising SEQ ID NO:8 or 23, or (b) HCDR regionsof SEQ ID NO:8 or 23 and remainder of the heavy chain variable regioncomprises more than 90% identity to SEQ ID NO:8 or 23; and (c) a lightchain variable region (LCVR) comprising SEQ ID NO:7 or 22, or (d) LCDRregions of SEQ ID NO:7 or 22 and remainder of the light chain variableregion comprises more than 90% identity to SEQ ID NO:7 or
 22. 11. Theantibody of claim 10, wherein the antibody comprises an HCVR comprisingSEQ ID NO:8 or HCDR regions of SEQ ID NO:8 and remainder of the heavychain variable region comprises more than 90% identity to SEQ ID NO:8,and an LCVR comprising SEQ ID NO:7 or LCDR regions of SEQ ID NO:7 andremainder of the light chain variable region comprises more than 90%identity to SEQ ID NO:7.
 12. The antibody of claim 10, wherein theantibody comprises an HCVR comprising SEQ ID NO:23 or HCDR regions ofSEQ ID NO:23 and remainder of the heavy chain variable region comprisesmore than 90% identity to SEQ ID NO:23, and an LCVR comprising SEQ IDNO:22 or LCDR regions of SEQ ID NO:22 and remainder of the light chainvariable region comprises more than 90% identity to SEQ ID NO:22.
 13. Anantibody comprising (a) a heavy chain comprising SEQ ID NO:12 or 13, or(b) HCDR regions of SEQ ID NO:12 or 13 and remainder of the heavy chaincomprises more than 90% identity to SEQ ID NO:12 or 13; and (c) a lightchain comprising SEQ ID NO:11, or (d) LCDR regions of SEQ ID NO:11 andremainder of the light chain variable region comprises more than 90%identity to SEQ ID NO:11.
 14. An antibody comprising (a) a heavy chaincomprising SEQ ID NO:27 or 28, or (b) HCDR regions of SEQ ID NO:27 or 28and remainder of the heavy chain comprises more than 90% identity to SEQID NO:27 or 28; and (c) a light chain comprising SEQ ID NO:26, or (d)LCDR regions of SEQ ID NO:26 and remainder of the light chain variableregion comprises more than 90% identity to SEQ ID NO:26.